Coelenterazine analogues and coelenteramide analogues

ABSTRACT

Coelenterazine analogues with different luminescence properties from conventional ones and coelenteramide analogues with different fluorescence properties from conventional ones have been desired. The invention provides coelenterazine analogues modified at the 8-position of coelenterazine and coelenteramide analogues modified at the 2- or 3-position of coelenteramide.

TECHNICAL FIELD

The present invention relates to coelenterazine analogue, coelenteramideanalogue, calcium-binding photoprotein, fluorescent protein, and thelike.

BACKGROUND ART

A calcium-binding photoprotein is one of the proteins responsible forbioluminescence. This photoprotein emits light upon specific interactionwith Ca²⁺. The calcium-binding photoprotein is a complex of a proteinhaving the catalytic function of oxygenation and a peroxide of luciferinas a light emitting substrate. In the calcium-binding photoprotein, theprotein having the catalytic function of oxygenation refers to as anapoprotein. The peroxide of a luciferin is coelenterazine peroxide(2-hydroperoxycoelenterazine). The calcium-binding photoproteinincluding aequorin, clytin-1, clytin-II, mitrocomin, obelin, etc. areknown and present in coelenterates. Among these photoproteins, aequorinis a photoprotein isolated from the luminous jellyfish Aequorea aequorea(1: Shimomura, In: Bioluminescence, Chemical Principles and Methods,(2006) pp 90-158, World Scientific Pub. Co.; 2: Shimomura et al., (1962)J. Cell. Comp. Physiol. 59, pp 223-240). Aequorin is a non-covalentcomplex of apoaequorin (21.4 kDa), which is an apoprotein, and ahydroperoxide of coelenterazine (3: Head et al., (2000) Nature, 405372-376). Apoaequorin is composed of 189 amino acid residues in a singlepolypeptide chain and has three EF-hand motifs characteristic ofCa²⁺-binding sites (4: Inouye et al., (1985) Proc. Natl. Acad Sci. USA.82, 3154-3158). In the presence of Ca²⁺, aequorin emits blue light(λ_(max)=˜460 nm) by an intramolecular reaction and decomposes itselfinto apoaequorin, coelenteramide and CO₂ (5: Shimomura & Johnson (1972)Biochemistry 11, 1602-1608.; 6: Shimomura & Johnson (1973) TetrahedronLeft. 2963-2966). The complex of Ca²⁺-binding apoaequorin withcoelenteramide obtained by this decomposition is known as bluefluorescent protein (BFP) (7: Shimomura & Johnson (1975) Nature 256,236-238). The fluorescence and luminescence spectra of this BFP areidentical to the bioluminescence spectra of aequorin (8: Shimomura(1995) Biochem. J. 306, 537-543; 9: Inouye (2004) FEBS Lett. 577,105-110).

Recombinant aequorin can be obtained by incubating recombinantapoaequorin prepared from Escherichia coli with coelenterazine in thepresence of EDTA and a reducing reagent (10: Inouye et al. (1986)Biochemistry 25, 8425-8429; 11: Inouye et al. (1989) J. Biochem. 105,473-477). This recombinant aequorin is highly purified (12: Shimomura &Inouye (1999) Protein Express. Purif. 16, 91-95). The luminescenceproperties of recombinant aequorin are identical to that of nativeaequorin (13: Shimomura et al. (1990) Biochem. J. 270, 309-312).

Approximately 50 types of coelenterazine analogues (CTZ analogues) werehitherto synthesized and some of them were actually used to preparesemi-synthetic aequorins (e.g., 13: Shimomura et al. (1990) Biochem. J.270, 309-312, 14: Shimomura O. et al. (1988) Biochem. J. 251, 405-410,15: Shimomura O. et al. (1989) Biochem. J. 261, 913-920, 16: Inouye S. &Shimomura O. (1997) Biochem. Biophys. Res. Commun. 233, 349-353).

The crystal structures of aequorin and semi-synthetic aequorins havebeen determined (3: Head et al. (2000) Nature 405, 372-376; 17: Toma etal. (2005) Protein Science 14, 409-416), and the binding properties ofMg²⁺ to EF-hand motif of aequorin were also investigated by NMR analysis(18: Ohashi et al. (2005) 1 J. Biochem. 38, 613-620).

Recently, BFP was quantitatively prepared from the purified recombinantaequorin (9: Inouye, FEBS Lett. 577 (2004) 105-110; 19: Inouye & Sasaki,FEBS Lett. 580 (2006) 1977-1982). BFP was found to have a substantialluminescence activity, catalyzing the oxidation of coelenterazine like aluciferase. The luminescence activity of BFP is about 10 times higherthan that of Ca²⁺-binding apoaequorin (9: Inouye, FEBS Lett. 577 (2004)105-110). Thus, BFP is a novel bifunctional protein having bothfluorescence and luciferase activities. BFP is further converted intogreen fluorescent protein (gFP) having the fluorescence emission maximumpeak at around 470 nm by the treatment of EDTA.

gFP is a non-covalent complex of apoaequorin with coelenteramide andaequorin can be obtained by incubation of gFP with coelenterazine at 25°C. in the absence of a reducing reagent (9: Inouye (2004) FEBS Lett.577, 105-110). By incubation of BFP or gFP with various coelenterazineanalogues in the presence of EDTA and dithiothreitol (DTT),semi-synthetic aequorins could be also prepared (19: Inouye & Sasaki(2006) FEBS Lett. 580, 1977-1982). Furthermore, the luminescenceactivity of BFP as a luciferase is stimulated by the addition ofimidazole at the concentrations of 30 to 300 mM using coelenterazine andits analogue as a substrate (20: Inouye & Sasaki (2007) Biochem.Biophys. Res. Commun. 354, 650-655).

In the development of use of BFP and gFP, there are some problems thatthe catalytic domain for oxygenation of coelenterazine, which isimportant basic information, or amino acid residues in BFP and gFP stillremain to be elucidated. In order to solve these problems and in thedevelopment of use, it is necessary to easily prepare several tensmilligrams of BFP and gFP and succeed. The present inventors have alsoestablished a method for preparing semi-synthetic gFP and semi-syntheticBFP from apoaequorin and chemically synthesized coelenteramide (21:Inouye & Hosoya (2009) Biochem. Biophys. Res. Commun. 386, 617-622).Semi-synthetic BFP prepared by this method shows a luciferase activityusing coelenterazine as a substrate, similar to BFP prepared fromaequorin by Ca2+-triggered luminescence reaction. The emission spectrumof semi-synthetic BFP is blue with around 470 nm.

When/As an in vivo probe to be used, a probe having the maximumwavelength of fluorescence closer to that of the near infrared region(longer than 600 nm) has been desired. In semi-synthetic BFP andsemi-synthetic gFP prepared from coelenteramide analogues andapoaequorin from native aequorin, however, those having a maximumwavelength of fluorescence at 485 nm or longer have not yet beenreported.

REFERENCES

-   1: Shimomura, In: Bioluminescence, Chemical principles and    methods (2006) pp 90-158, World Scientific Pub. Co.-   2: Shimomura et al. (1962) J. Cell. Comp. Physiol. 59, 223-240-   3: Head et al. (2000) Nature 405, 372-376-   4: Inouye et al. (1985) Proc. Natl. Acad Sci. USA. 82, 3154-3158-   5: Shimomura & Johnson (1972) Biochemistry 11, 1602-1608-   6: Shimomura & Johnson (1973) Tetrahedron Lett. 2963-2966-   7: Shimomura & Johnson (1975) Nature 256, 236-238-   8: Shimomura (1995) Biochem. J. 306, 537-543-   9: Inouye (2004) FEBS Lett. 577, 105-110-   10: Inouye et al (1986) Biochemistry 25, 8425-8429-   11: Inouye et al (1989) J. Biochem. 105 473-477-   12: Shimomura & Inouye (1999) Protein Express. Purif. 16, 91-95-   13: Shimomura et al. (1990) Biochem. J. 270, 309-312-   14: Shimomura et al. (1988) Biochem. J. 251, 405-410-   15: Shimomura et al. (1989) Biochem. J. 261, 913-920-   16: Inouye S. & Shimomura O. (1997) Biochem. Biophys. Res. Commun.    233, 349-353-   17: Toma et al. (2005) Protein Science 14, 409-416-   18: Ohashi et al. (2005) J. Biochem. 138, 613-620-   19: Inouye & Sasaki (2006) FEBS Lett. 580, 1977-1982-   20: Inouye & Sasaki (2007) Biochem. Biophys. Res. Commun. 354,    650-655-   21: Inouye & Hosoya (2009) Biochem. Biophys. Res. Commun. 386,    617-622

DISCLOSURE OF INVENTION

Under the foregoing circumstances, coelenterazine analogues showingdifferent luminescence properties from conventional ones have also beendesired.

Furthermore, coelenteramide analogues showing different fluorescenceproperties from conventional ones and fluorescent proteins containingsuch coelenteramide analogues have been desired.

In order to solve the above problems, the present inventors have madeextensive investigations and, as a result, have found thatcoelenterazine analogues modified at the 8-position of coelenterazineshow different luminescence properties from those of known compounds,that coelenteramide analogues modified at the 2- or 3-position ofcoelenteramide show different luminescence properties from those ofknown compounds, and so on. Thus, the present invention has come to beaccomplished.

The present invention provides the following coelenterazine analogues,coelenteramide analogues, calcium-binding photoproteins, fluorescentproteins, and so on.

[1]A compound represented by general formula (I) shown below:

wherein R³ is hydrogen atom, bromine atom and any one selected from thegroups represented by formulas below:

wherein each of R⁶, R⁷ and R⁸ independently represents hydrogen atom, asubstituted or unsubstituted alkyl having 1 to 6 carbon atoms, asubstituted or unsubstituted aryl, or a substituted or unsubstitutedheteroaryl; and R⁶ and R⁸ may be combined together to form a substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl withthe carbon atom bound to each of R⁶ and R⁸.

[2] The compound according to [1] above, which is selected from thecompounds shown below.

[3] The compound according to [1] above, which is selected from thecompounds shown below.

[4] A compound represented by general formula (II) below:

wherein R^(3′) is hydrogen atom, bromine atom and any one selected fromthe groups represented by formulas below:

wherein each of R^(6′), R^(7′) and R^(8′) independently representshydrogen atom, a substituted or unsubstituted alkyl having 1 to 6 carbonatoms, a substituted or unsubstituted aryl, or a substituted orunsubstituted heteroaryl; and R^(6′) and R^(8′) may be combined togetherto form a substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl with the carbon atom bound to each of R^(6′)and R^(8′).

[5] The compound according to [4] above, which is selected from thecompounds shown below.

[6] A compound represented by general formula (III) below:

wherein Z¹ is O or S; and R^(3″) is hydrogen atom, bromine atom, asubstituted or unsubstituted aryl, a substituted or unsubstitutedarylalkyl, a substituted or unsubstituted arylalkenyl, a substituted orunsubstituted arylalkynyl, an alkyl which may optionally be substitutedwith an alicyclic group, an alkenyl which may optionally be substitutedwith an alicyclic group, an alkynyl which may optionally be substitutedwith an alicyclic group, an alicyclic group, or a heterocyclic group.

[7]A compound represented by general formula (IV) below:

wherein R^(2′″) is a group selected from the groups shown below:

and R^(3′″) is hydrogen atom, bromine atom, a substituted orunsubstituted aryl, a substituted or unsubstituted arylalkyl, asubstituted or unsubstituted arylalkenyl, a substituted or unsubstitutedarylalkynyl, an alkyl which may optionally be substituted with analicyclic group, an alkenyl which may optionally be substituted with analicyclic group, an alkynyl which may optionally be substituted with analicyclic group, an alicyclic group, or a heterocyclic group.

[8] The compound according to [6] or [7] above, which is selected fromthe compounds shown below.

[9]A calcium-binding photoprotein comprising a peroxide of the compoundaccording to any one of [1] to [3] above and an apoprotein of acalcium-binding photoprotein.

[10]A process for producing a calcium-binding photoprotein, whichcomprises contacting the compound according to any one of [1] to [3]above with an apoprotein of a calcium-binding photoprotein to obtain thecalcium-binding photoprotein.

[11]A method for detecting or quantifying calcium ions, which comprisesusing the calcium-binding photoprotein according to [9] above.

[12]A method for analyzing a physiological function or enzyme activity,which comprises performing the bioluminescence resonance energy transfer(BRET) method using the calcium-binding photoprotein according to [9]above as a donor protein.

[13]A fluorescent protein comprising the compound according to any oneof [4] to [8] above, an apoprotein of a calcium-binding photoprotein andcalcium ions or divalent or trivalent ions replaceable for the calciumions.

[14]A method for producing a fluorescent protein, which comprisescontacting the calcium-binding photoprotein according to [9] above withcalcium ions or divalent or trivalent ions replaceable for the calciumions to obtain the fluorescent protein.

[15]A method for producing a fluorescent protein, which comprisescontacting the compound according to any one of [4] to [8] above with anapoprotein of a calcium-binding photoprotein in the presence of calciumions or divalent or trivalent ions replaceable for the calcium ions toobtain the fluorescent protein.

[16] The method according to [14] or [15] above, wherein the contact iscarried out in the presence of a reducing agent.

[17]A fluorescent protein comprising the compound according to any oneof [4] to [8] above and an apoprotein of a calcium-binding photoprotein.

[18]A method for producing a fluorescent protein, which comprisescontacting the compound according to any one of [4] to [8] above with anapoprotein of a calcium-binding photoprotein in the presence of achelating agent for removing calcium ions or divalent or trivalent ionsreplaceable for the calcium ions to obtain the fluorescent protein.

[19]A method for producing a fluorescent protein, which comprisestreating the fluorescent protein according to [13] above with achelating agent for removing calcium ions or divalent or trivalent ionsreplaceable for the calcium ions.

[20] The method according to [18] or [19] above, wherein the contact iscarried out in the presence of a reducing agent.

[21]A method for analyzing a physiological function or enzyme activity,which comprises performing the fluorescence resonance energy transfer(FRET) method using the fluorescent protein according to [13] or [17]above as an acceptor or a donor.

According to the present invention, a novel coelenterazine analogue isprovided. Coelenterazine analogues in some embodiments of the presentinvention show different luminescence properties from those of knowncoelenterazine.

According to the present invention, a novel coelenteramide analogue isprovided. Coelenteramide analogues in some embodiments of the presentinvention show different fluorescence properties from those of knowncoelenteramide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the luminescence spectra of semi-synthetic aequorinsprepared from coelenterazine analogues by addition of calcium.

FIG. 2 shows the fluorescence spectra of fluorescent proteins obtainedby adding calcium ions to semi-synthetic aequorins prepared fromcoelenterazine analogues.

FIG. 3 shows the relationship between the initial luminescenceintensities of semi-synthetic aequorins and various concentrations ofcalcium ion.

FIG. 4 shows the fluorescence spectra of semi-synthetic gFP preparedfrom coelenteramide analogues (4a to 4l) and apoaequorin in the presenceof EDTA.

FIG. 5 shows the fluorescence spectra of semi-synthetic gFP preparedfrom coelenteramide analogues (4m to 4z) and apoaequorin in the presenceof EDTA.

FIG. 6 shows the fluorescence spectra of semi-synthetic BFP preparedfrom coelenteramide analogues (4a to 4l) and apoaequorin in the presenceof calcium ions.

FIG. 7 shows the fluorescence spectra of semi-synthetic BFP preparedfrom coelenteramide analogues (4m to 4z) and apoaequorin in the presenceof calcium ions.

FIG. 8 shows the scheme for preparing BFP, gFP, aequorin, etc. fromcoelenteramide.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

1. Coelenterazine Analogues of the Invention

The present invention provides the compounds represented by generalformula (I) (sometimes referred to as the “coelenterazine analogues ofthe present invention”). Coelenterazine analogues are modified at theC-8 position of coelenterazine.

(wherein R³ is hydrogen atom, bromine atom and any one selected from thegroups represented by formulas below:

wherein each of R⁶, R⁷ and R⁸ independently represents hydrogen atom, asubstituted or unsubstituted alkyl having 1 to 6 carbon atoms, asubstituted or unsubstituted aryl, or a substituted or unsubstitutedheteroaryl; and R⁶ and R⁸ may be combined together to form a substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl withthe carbon atom bound to each of R⁶ and R⁸).

As used herein, the “substituted or unsubstituted alkyl having 1 to 6carbon atoms” shown by R⁶, R⁷ and R⁸ is, for example, an alkyl having 1to 6 carbon atoms and carrying 1 to 5 substituents which may be the sameor different, or an unsubstituted alkyl. Examples of the substituentinclude at least one selected from the group consisting of a halogen(fluorine, chlorine, bromine or iodine), hydroxy group, nitro, cyano,amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl,neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to 6 carbonatoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isopropoxy,sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy, t-pentoxy,hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbon atoms (e.g.,dimethylamino, diethylamino, ethylmethylamino, diisopropylamino,1-piperidinyl, 1-pyrrolidinyl), and the like. The “substituted orunsubstituted alkyl having 1 to 6 carbon atoms” is methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,isopentyl, neopentyl, t-pentyl, hexyl, isohexyl, fluoromethyl, aperfluoroalkyl (e.g., trifluoromethyl, perfluorohexyl), or the like.

The “substituted or unsubstituted aryl” shown by R⁶, R⁷ and R⁸ or the“substituted or unsubstituted aryl” formed by combining R⁶ and R⁸together with the carbon atom bound to each of R⁶ and R⁸ is, forexample, an aryl having 1 to 5 substituents, which are the same ordifferent, or an unsubstituted aryl. The substituent includes, forexample, at least one selected from the group consisting of a halogen(fluorine, chlorine, bromine or iodine), hydroxy group, nitro, cyano,amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl,neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to 6 carbonatoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy, t-pentoxy,hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbon atoms (e.g.,dimethylamino, diethylamino, ethylmethylamino, diisopropylamino,1-piperidinyl, 1-pyrrolidinyl), and the like. In some embodiments of thepresent invention, the substituent includes hydroxy group, nitro ordimethylamino. The “substituted or unsubstituted aryl” specificallyincludes phenyl, a naphthyl (1-naphthyl, 2-naphthyl), a hydroxyphenyl(2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl), an aminophenyl(e.g., 4-dimethylaminophenyl), a nitrophenyl (e.g., 4-nitrophenyl), afluorophenyl (e.g., 4-fluorophenyl), a trifluoromethylphenyl (e.g.,4-trifluoromethylphenyl), and the like.

The “substituted or unsubstituted heteroaryl” shown by R⁶, R⁷ and R⁸ orthe “substituted or unsubstituted heteroaryl” formed by combining R⁶ andR⁸ together with the carbon atom bound to each of R⁶ and R⁸ is, forexample, a heteroaryl having 1 to 5 substituents, which are the same ordifferent, or an unsubstituted heteroaryl. The substituent includes, forexample, at least one selected from the group consisting of a halogen(fluorine, chlorine, bromine or iodine), hydroxy group, nitro, cyano,amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl,neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to 6 carbonatoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy, t-pentoxy,hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbon atoms (e.g.,dimethylamino, diethylamino, ethylmethylamino, diisopropylamino,1-piperidinyl and 1-pyrrolidinyl, preferably, dimethylamino,diethylamino, ethylmethylamino and diisopropylamino), an aryl (e.g.,phenyl, naphthyl, anthranyl), a heteroaryl (e.g., a thienyl (2-thienyl,3-thienyl), a furyl (2-furyl, 3-furyl), a pyrrolyl (2-pyrrolyl,3-pyrrolyl), a pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), imidazoyl,pyrazoyl, oxazoyl, thiazoyl, isoxazoyl, pyrimidyl, pyrazyl, pyridazoyl),and the like. Specifically, the “substituted or unsubstitutedheteroaryl” is a thienyl (2-thienyl, 3-thienyl), a benzothienyl (e.g.,benzo[b]thiophen-2-yl, benzo[b]thiophen-3-yl), a thienothienyl (e.g.,thieno[2,3-b]thienyl, thieno[3,2-b]thienyl, thieno[3,2-b:2,3-d]thienyl),a bithiophenyl (e.g., 2,2-bithiophen-5-yl), benzofuranyl, indoyl,indazoyl, benzimidazoyl, benzoxazoyl, benzisoxazoyl, quinolinyl,isoquinolinyl, quinoxalinyl, quinazolinyl, acridinyl, or the like.

In a preferred embodiment of the present invention, coelenterazineanalogue includes the following compounds.

In a more preferred embodiment of the present invention, coelenterazineanalogue includes the following compounds.

The calcium-binding photoprotein which contains some coelenterazineanalogues of the present invention as a light emitting substrateprovides a longer half decay time of luminescence, which is a timeperiod in which the luminescence becomes half of the maximumluminescence intensity, when compared to a photoprotein containingcoelenterazine as a light emitting substrate. For example, the halfdecay time is longer by 1.1 times or more, 1.2 times or more, 1.3 timesor more, 1.4 times or more, 1.5 times or more, 2.0 times or more, 3.0times or more, 4.0 times or more, 5.0 times or more, 10 times or more,15 times or more, 20 times or more, 30 times or more, 40 times or more,50 times or more, 60 times or more, or 70 times or more. Morespecifically, coelenterazine analogues, which can provide a longer halfdecay time of luminescence than that of coelenterazine, are 3a, 3b, 3d,3e, 3f, 3h and 3j, preferably 3a, 3e, 3f and 3j and more preferably 3f,in coelenterazine analogues (3a to 3t).

The calcium-binding photoprotein which contains some more coelenterazineanalogues of the present invention as a light emitting substrate givesdifferent luminescence spectra, unlike fluorescence spectra of thecorresponding fluorescent protein prepared by generating luminescence ofthe calcium-binding photoprotein by addition of calcium. For example,the maximum emission wavelength between the luminescence and thefluorescence is different by ±20 nm or more, ±25 nm or more, ±30 nm ormore, ±35 nm or more, ±40 nm or more, ±45 nm or more, ±50 nm or more,±60 nm or more, ±70 nm or more, ±80 nm or more, ±90 nm or more, ±100 nmor more, ±110 nm or more, ±120 nm or more, or ±130 nm. Morespecifically, coelenterazine analogues, which can provide differentluminescence spectra and fluorescence spectra, are 3a, 3b, 3e, 3f, 3hand 3j, preferably 3a, 3e and 3j and more preferably 3b and 3j, incoelenterazine analogues (3a to 3t).

2. Method for Producing Coelenterazine Analogue of the Invention

Coelenterazine analogue of the present invention described above, i.e.,coelenterazine analogue represented by general formula (I) below.

(wherein R³ is the same as defined above) can be produced as follows.That is, coelenterazine analogue represented by general formula (I) canbe obtained by reacting a compound represented by general formula (I-1)below:

(wherein R³ is the same as defined above and Y¹ is hydrogen atom or aprotecting group) with a compound represented by general formula (I-2)below:

(wherein Y² is hydrogen atom or a protecting group, R² is independentlymethyl, ethyl, propyl, butyl or isopropyl, and two R² may be combinedtogether to form an alkylene having 2 to 4 carbon atoms). Thus, thecompound represented by general formula (I) can be obtained.

As used herein, the protecting groups shown by Y¹ and Y² independentlyrepresent, e.g., tert-butyldimethylsilyl (TBDMS),tert-butyldiphenylsilyl (TBDPS), trimethylsilyl (TMS), triethylsilyl(TES), triisopropylsilyl (TIPS), methoxymethyl (MOM),2-methoxethoxyymethyl (MEM), 2-tetrahydropyranyl (THP), etc.,particularly preferably TBDMS, MOM or THP.

The alkylene having 2 to 4 carbon atoms, which are formed by combiningtwo R² together, includes ethylene, propylene, butylene, etc.

The compound represented by general formula (I-1) can be produced byknown methods. For example, the compound represented by general formula(I-1) can be produced by, for example, the methods described in Y. Kishiet al., Tetrahedron Lett., 13, 2747-2748 (1972), M. Adamczyk et al.,Org. Prep. Proced Int., 33, 477-485 (2001) or F. D. Wael et al., Bioorg.Med Chem., 17, 4336-4344 (2009), or modifications of these methods. Morespecifically, the compound represented by general formula (I-1) can beproduced as follows. First, cyclization of a substituted phenylglyoxalaldoxime and a glycinonitrile derivative is carried out to form thepyrazine oxide. Subsequently, the pyrazine oxide is subjected tocatalytic hydrogenation using Raney Ni, etc. as a catalyst to producethe compound. Alternatively, the compound can be produced either by theSuzuki-Miyaura coupling reaction of a 2-amino-5-bromopyrazine derivativehaving a suitable substituent at the 3-position and a substituted phenylborate or a substituted pinacol borate ester, or by the Suzuki-Miyauracoupling reaction of a 2-amino-5-bromopyrazine derivative and a suitableboric acid derivative or a pinacol borate ester.

The compound represented by general formula (I-2) can be produced byknown methods. For example, the compound represented by general formula(I-2) can be produced by, for example, the methods described in M.Adamczyk, M. et al., Synth. Commun., 32, 3199-3205 (2002) or H. Baganz &H.-J. May, Chem. Ber, 99, 3766-3770 (1966) and H. Baganz & H.-J. May,Angew. Chem. Int. Ed. Eng., 5, 420 (1966), or modifications of thesemethods. More specifically, the compound represented by general formula(I-2) can be produced by reacting a substituted benzyl Grignard reagentwith ethyl diethoxyacetate at a low temperature (−78° C.), or byreacting an α-diazo-α-substituted phenyl ketone with tert-butylhypochlorite in ethanol.

As used herein, the reagent used in the method for producing thecompound of the present invention represented by general formula (I) isan acid catalyst such as hydrochloric acid, hydrobromic acid, hydroiodicacid, hydrofluoric acid, perchloric acid, sulfuric acid, phosphoricacid, nitric acid, acetic acid, etc. These reagents may be used alone oras a mixture thereof; hydrochloric acid is particularly preferred.

The solvent used in the method for producing the compound of the presentinvention represented by general formula (I) is not particularlylimited, but various solvents can be used. Examples of the solvent aredioxane, tetrahydrofuran, ether, methanol, ethanol, water, etc., whichcan be used alone or as a mixture thereof.

In the method for producing the compound of the present inventionrepresented by general formula (I), the reaction temperature andreaction time are not particularly limited but are generally at 0° C. to200° C. for 1 to 96 hours, at room temperature to 150° C. for 3 to 72hours, or at 60° C. to 120° C. for 6 to 24 hours.

3. Coelenteramide Analogue of the Invention

The present invention provides the compound represented by generalformula (II) or general formula (III) below (hereinafter sometimesreferred to as “coelenteramide analogue of the present invention”).Coelenteramide analogue of the present invention is modified at the C-3or C-2 position of coelenteramide.

3.1. Coelenteramide Analogue Modified at the C-3 Position

Coelenteramide analogue of the present invention, which is modified atthe 3-position, includes the following compounds.

(wherein R^(3′) is hydrogen atom, bromine atom and any one selected fromthe groups represented by formulas below:

wherein each of R^(6′), R^(7′) and R^(8′) independently representshydrogen atom, a substituted or unsubstituted alkyl having 1 to 6 carbonatoms, a substituted or unsubstituted aryl, or a substituted orunsubstituted heteroaryl; and R^(6′) and R^(8′) may be combined togetherto form a substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl with the carbon atom bound to each of R^(6′)and R^(8′)).

As used herein, the “substituted or unsubstituted alkyl having 1 to 6carbon atoms” shown by R^(6′), R^(7′) and R^(8′) is, for example, analkyl having 1 to 6 carbon atoms and carrying 1 to 5 substituents whichare the same or different, or an unsubstituted alkyl. Examples of thesubstituent include at least one selected from the group consisting of ahalogen (fluorine, chlorine, bromine or iodine), hydroxy group, nitro,cyano, amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,isopentyl, neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to6 carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy,t-pentoxy, hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbonatoms (e.g., dimethylamino, diethylamino, ethylmethylamino,diisopropylamino, 1-piperidinyl, 1-pyrrolidinyl), and the like. The“substituted or unsubstituted alkyl having 1 to 6 carbon atoms” ismethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl,pentyl, isopentyl, neopentyl, t-pentyl, hexyl, isohexyl, fluoromethyl, aperfluoroalkyl (e.g., trifluoromethyl, perfluorohexyl), or the like.

As used herein, the “substituted or unsubstituted aryl” shown by R⁶,R^(7′) and R^(8′) or the “substituted or unsubstituted aryl” formed bycombining R⁶, and R^(8′) together with the carbon atom bound to each ofR^(6′) and R^(8′) is, for example, an aryl having 1 to 5 substituentswhich are the same or different, or an unsubstituted aryl. Examples ofthe substituent include at least one selected from the group consistingof a halogen (fluorine, chlorine, bromine or iodine), hydroxy group,nitro, cyano, amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,isopentyl, neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to6 carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy,t-pentoxy, hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbonatoms (e.g., dimethylamino, diethylamino, ethylmethylamino,diisopropylamino, 1-piperidinyl, 1-pyrrolidinyl) and the like. In someembodiments of the present invention, the substituent is hydroxy group,nitro or dimethylamino. Specifically, the “substituted or unsubstitutedaryl” includes phenyl, a naphthyl (1-naphthyl, 2-naphthyl), ahydroxyphenyl (2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl), anaminophenyl (e.g., 4-dimethylaminophenyl), a nitrophenyl (e.g.,4-nitrophenyl), a fluorophenyl (e.g., 4-fluorophenyl), atrifluoromethylphenyl (e.g., 4-trifluoromethylphenyl), etc.

The “substituted or unsubstituted heteroaryl” shown by R^(6′), R^(7′)and R^(8′) or the “substituted or unsubstituted heteroaryl” formed bycombining R^(6′) and R^(8′) together with the carbon atom bound to eachof R^(6′) and R^(8′) is, for example, a heteroaryl having 1 to 5substituents which are the same or different, or an unsubstitutedheteroaryl Examples of the substituent include at least one selectedfrom the group consisting of a halogen (fluorine, chlorine, bromine oriodine), hydroxy group, nitro, cyano, amino, an alkyl having 1 to 6carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl,isohexyl), an alkoxyl having 1 to 6 carbon atoms (e.g., methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy,isopentoxy, neopentoxy, t-pentoxy, hexyloxy, isohexyloxy), adialkylamino having 1 to 6 carbon atoms (e.g., dimethylamino,diethylamino, ethylmethylamino, diisopropylamino, 1-piperidinyl and1-pyrrolidinyl, preferably, dimethylamino, diethylamino,ethylmethylamino, diisopropylamino), an aryl (e.g., phenyl, naphthyl,anthranyl), a heteroaryl (e.g., a thienyl (2-thienyl, 3-thienyl), afuryl (2-furyl, 3-furyl), a pyrrolyl (2-pyrrolyl, 3-pyrrolyl), a pyridyl(2-pyridyl, 3-pyridyl, 4-pyridyl), imidazoyl, pyrazoyl, oxazoyl,thiazoyl, isoxazoyl, pyrimidyl, pyrazyl, pyridazoyl) and the like.Specifically, the “substituted or unsubstituted heteroaryl” is a thienyl(2-thienyl, 3-thienyl), a benzothienyl (e.g., benzo[b]thiophen-2-yl,benzo[b]thiophen-3-yl), a thienothienyl (e.g., thieno[2,3-b]thienyl,thieno[3,2-b]thienyl, thieno[3,2-b:2,3-d]thienyl), a bithiophenyl (e.g.,2,2-bithiophen-5-yl), benzofuranyl, indoyl, indazoyl, benzimidazoyl,benzoxazoyl, benzisoxazoyl, quinolinyl, isoquinolinyl, quinoxalinyl,quinazolinyl, acridinyl, etc.

In a preferred embodiment of the present invention, coelenteramideanalogue modified at the C-3 position includes the following compounds.

3.2. Coelenteramide Analogue Modified at the C-2 Position

Coelenteramide analogue in some embodiment of the present invention,which is modified at the C-2-position, includes the following compounds.

(wherein Z¹ is O or S; and R^(3″) is hydrogen atom, bromine atom, asubstituted or unsubstituted aryl, a substituted or unsubstitutedarylalkyl, a substituted or unsubstituted arylalkenyl, a substituted orunsubstituted arylalkynyl, an alkyl which may optionally be substitutedwith an alicyclic group, an alkenyl which may optionally be substitutedwith an alicyclic group, an alkynyl which may optionally be substitutedwith an alicyclic group, an alicyclic group, or a heterocyclic group.)

As used herein, the “substituted or unsubstituted aryl” shown by R^(3″)is, for example, an aryl having 1 to 5 substituents, or an unsubstitutedaryl. For example, the substituted or unsubstituted aryl is at least oneselected from the group consisting of a halogen (fluorine, chlorine,bromine or iodine), hydroxy group, an alkyl having 1 to 6 carbon atoms(e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl, isohexyl), analkoxyl having 1 to 6 carbon atoms (e.g., methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy,isopentoxy, neopentoxy, t-pentoxy, hexyloxy, isohexyloxy), amino, adialkylamino having 1 to 6 carbon atoms (e.g., dimethylamino,diethylamino, ethylmethylamino, diisopropylamino, 1-piperidinyl,1-pyrrolidinyl), and the like. In some embodiments of the presentinvention, the substituent is hydroxy group. Specifically, the“substituted or unsubstituted aryl” is phenyl, p-hydroxyphenyl,p-aminophenyl, p-dimethylaminophenyl, or the like, preferably phenyl,p-hydroxyphenyl, etc. In some embodiments of the present invention, the“substituted or unsubstituted aryl” is an unsubstituted aryl, e.g.,phenyl, etc.

The “substituted or unsubstituted arylalkyl” shown by R^(3″) is, forexample, an arylalkyl having 7 to 10 carbon atoms and carrying 1 to 5substituents, or an unsubstituted arylalkyl having 7 to 10 carbon atoms.Examples of the substituents include a halogen (fluorine, chlorine,bromine or iodine), hydroxy group, an alkyl having 1 to 6 carbon atoms(e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl, isohexyl), analkoxyl having 1 to 6 carbon atoms (e.g., methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy,isopentoxy, neopentoxy, t-pentoxy, hexyloxy, isohexyloxy), amino, adialkylamino having 1 to 6 carbon atoms (e.g., dimethylamino,diethylamino, ethylmethylamino, diisopropylamino, 1-piperidinyl,1-pyrrolidinyl), and the like. The “substituted or unsubstitutedarylalkyl” includes, for example, benzyl, α-hydroxybenzyl, phenylethyl,p-hydroxybenzyl, p-dimethylaminobenzyl, etc., preferably benzyl,α-hydroxybenzyl, phenylethyl, etc. In some embodiments of the presentinvention, the “substituted or unsubstituted arylalkyl” is benzyl.

The “substituted or unsubstituted arylalkenyl” shown by R^(3″) is, forexample, an arylalkenyl having 8 to 10 carbon atoms and carrying 1 to 5substituents, or an unsubstituted arylalkenyl having 8 to 10 carbonatoms. Examples of the substituents include a halogen (fluorine,chlorine, bromine or iodine), hydroxy group, an alkyl having 1 to 6carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl,isohexyl), an alkoxyl having 1 to 6 carbon atoms (e.g., methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy,isopentoxy, neopentoxy, t-pentoxy, hexyloxy, isohexyloxy), amino, adialkylamino having 1 to 6 carbon atoms (e.g., dimethylamino,diethylamino, ethylmethylamino, diisopropylamino, 1-piperidinyl,1-pyrrolidinyl), and the like. For example, the “substituted orunsubstituted arylalkenyl” is phenylvinyl, p-hydroxyphenylvinyl, orp-dimethylaminophenylvinyl. In some embodiments of the presentinvention, the “substituted or unsubstituted arylalkenyl” is anunsubstituted arylalkenyl, e.g., phenylvinyl.

The “substituted or unsubstituted arylalknyl” shown by R^(3″) is, forexample, an arylalknyl having 8 to 10 carbon atoms and carrying 1 to 5substituents, or an unsubstituted arylalknyl having 8 to 10 carbonatoms. Examples of the substituents include a halogen (fluorine,chlorine, bromine or iodine), hydroxy group, an alkyl having 1 to 6carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl,isohexyl), an alkoxyl having 1 to 6 carbon atoms (e.g., methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy,isopentoxy, neopentoxy, t-pentoxy, hexyloxy, isohexyloxy), amino, adialkylamino having 1 to 6 carbon atoms (e.g., dimethylamino,diethylamino, ethylmethylamino, diisopropylamino, 1-piperidinyl,1-pyrrolidinyl), and the like. Examples of the “substituted orunsubstituted arylalknyl” are phenylethynyl, naphthylethynyl,4-fluorophenylethynyl, 4-trifluoromethylphenylethynyl,4-methoxyphenylethynyl, 4-nitrophenylethynyl, etc., preferably,phenylethynyl, etc.

The “alkyl which may optionally be substituted with an alicyclic group”shown by R^(3″) is, for example, an unsubstituted straight or branchedalkyl having 1 to 4 carbon atoms, or a straight or branched alkyl having1 to 4 carbon atoms, which is substituted with an alicyclic grouphaving, e.g., 1 to 10 carbon atoms. Examples of the alicyclic groupinclude cyclohexyl, cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, andthe like. Preferably, the alicyclic group is cyclohexyl, cyclopentyl oradamantyl. Examples of the “alkyl which may optionally be substitutedwith an alicyclic group” are methyl, ethyl, propyl, 2-methylpropyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,cyclobutylmethyl, cyclopropylmethyl, or the like, preferably, methyl,ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl,cyclohexylmethyl, cyclohexylethyl, etc. In some embodiments of thepresent invention, the “alkyl which may optionally be substituted withan alicyclic group” is a straight alkyl which may optionally besubstituted with an alicyclic group, for example, methyl, ethyl, propyl,adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,etc.

The “alkenyl which may optionally be substituted with an alicyclicgroup” shown by R^(3″) is, for example, an unsubstituted straight orbranched alkenyl having 2 to 6 carbon atoms, or a straight or branchedalkenyl having 2 to 6 carbon atoms, which is substituted with analicyclic group having, e.g., 1 to 10 carbon atoms. Examples of thealicyclic group include cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, etc. Preferably, the alicyclic group is cyclohexyl,cyclopentyl, adamantly, or the like. The “alkenyl which may optionallybe substituted with an alicyclic group” includes, for example, vinyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,2-methylpropenyl, etc., preferably, 2-methylpropenyl, etc.

The “alkynyl which may optionally be substituted with an alicyclicgroup” shown by R^(3″) is, for example, an unsubstituted straight orbranched alkynyl having 2 to 6 carbon atoms, or a straight or branchedalkynyl having 2 to 6 carbon atoms, which is substituted with analicyclic group having, e.g., 1 to 10 carbon atoms. Examples of thealicyclic group include cyclohexyl, cyclopentyl, adamantyl, cyclobutyl,cyclopropyl, and the like. Preferably, the alicyclic group iscyclohexyl, cyclopentyl, adamantly, etc. Examples of the “alkynyl whichmay optionally be substituted with an alicyclic group” include ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, 5-hexynyl, and the like, preferably, 1-propynyl, etc.

The “aliphatic group” shown by R^(3″) includes, for example, cyclohexyl,cyclopentyl, adamantyl, cyclobutyl, cyclopropyl, and the like,preferably, cyclohexyl, etc.

The “heterocyclic group” shown by R^(3″) is, for example, a group formedby a 5- to 7-membered ring containing 1 to 3 atoms selected from thegroup consisting of N, O and S as the atoms for forming the ring, inaddition to carbons and bound through carbon, a group formed bycondensing at least two such rings and bound through carbon, or a groupformed by condensing such rings with a benzene ring and bound throughcarbon. Examples of the “heterocyclic group” are thiophen-2-yl,2-furanyl, 4-pyridyl, etc. In some embodiments of the present invention,the “heterocyclic group” is a heterocyclic group containing sulfur,e.g., thiophen-2-yl.

In a preferred embodiment of the present invention, R^(3′) is phenyl,p-hydroxyphenyl, benzyl, α-hydroxybenzyl, phenylethyl, phenylvinyl,cyclohexyl, cyclohexylmethyl, cyclohexylethyl, methyl, ethyl, propyl,2-methylpropyl, 2-methylpropenyl, adamantylmethyl, cyclopentylmethyl orthiophen-2-yl. In a more preferred embodiment of the present invention,R^(3″) is benzyl.

In a still preferred embodiment of the present invention, R^(3″) ishydrogen atom, bromine atom or any one selected from the groups shownbelow:

(wherein each of R^(6″), R^(7″) and R^(8″) independently representshydrogen atom, a substituted or unsubstituted alkyl having 1 to 6 carbonatoms, a substituted or unsubstituted aryl, or a substituted orunsubstituted heteroaryl, and R^(6″) and R^(8″) may be combined togetherwith the carbon atom bound to each of R^(6″) and R^(8″) to form asubstituted or unsubstituted aryl or a substituted or unsubstitutedheteroaryl).

As used herein, the “substituted or unsubstituted alkyl having 1 to 6carbon atoms” shown by R^(6″), R^(7″) and R^(8″) is, for example, analkyl having 1 to 6 carbon atoms and carrying 1 to 5 substituents whichare the same or different, or an unsubstituted alkyl. The substituentis, for example, at least one selected from the group consisting of ahalogen (fluorine, chlorine, bromine or iodine), hydroxy group, nitro,cyano, amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,isopentyl, neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to6 carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy,t-pentoxy, hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbonatoms (e.g., dimethylamino, diethylamino, ethylmethylamino,diisopropylamino, 1-piperidinyl, 1-pyrrolidinyl), etc. Specifically, the“substituted or unsubstituted alkyl having 1 to 6 carbon atoms” ismethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl,pentyl, isopentyl, neopentyl, t-pentyl, hexyl, isohexyl, fluoromethyl, aperfluoroalkyl (e.g., trifluoromethyl, perfluorohexyl), etc.

The “substituted or unsubstituted aryl” shown by R^(6″), R^(7″) andR^(8″) or the “substituted or unsubstituted aryl” formed by combiningR^(6″) and R^(8″) together with the carbon atom bound to each of R^(6″)and R^(8″) is, for example, an aryl having 1 to 5 substituents which arethe same or different, or an unsubstituted aryl. Examples of thesubstituent include at least one selected from the group consisting of ahalogen (fluorine, chlorine, bromine or iodine), hydroxy group, nitro,cyano, amino, an alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl,isopentyl, neopentyl, t-pentyl, hexyl, isohexyl), an alkoxyl having 1 to6 carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy,t-pentoxy, hexyloxy, isohexyloxy), a dialkylamino having 1 to 6 carbonatoms (e.g., dimethylamino, diethylamino, ethylmethylamino,diisopropylamino, 1-piperidinyl, 1-pyrrolidinyl), etc. In someembodiments of the present invention, the substituent is hydroxy group,nitro or dimethylamino. Specific examples are phenyl, a naphthyl(1-naphthyl, 2-naphthyl), a hydroxyphenyl (2-hydroxyphenyl,3-hydroxyphenyl, 4-hydroxyphenyl), an aminophenyl (e.g.,4-dimethylaminophenyl), a nitrophenyl (e.g., 4-nitrophenyl), afluorophenyl (e.g., 4-fluorophenyl), a trifluoromethylphenyl (e.g.,4-trifluoromethylphenyl), etc.

The “substituted or unsubstituted heteroaryl” shown by R^(6″), R^(7″)and R^(8″) or the “substituted or unsubstituted heteroaryl” formed bycombining R^(6″) and R^(8″) together with the carbon atom bound to eachof R^(6″) and R^(8″) is, for example, a heteroaryl having 1 to 5substituents which are the same or different, or an unsubstitutedheteroaryl. Examples of the substituent include at least one selectedfrom the group consisting of a halogen (fluorine, chlorine, bromine oriodine), hydroxy group, nitro, cyano, amino, an alkyl having 1 to 6carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl,isohexyl), an alkoxyl having 1 to 6 carbon atoms (e.g., methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t-butoxy, pentoxy,isopentoxy, neopentoxy, t-pentoxy, hexyloxy, isohexyloxy), adialkylamino having 1 to 6 carbon atoms (e.g., dimethylamino,diethylamino, ethylmethylamino, diisopropylamino, 1-piperidinyl and1-pyrrolidinyl, preferably, dimethylamino, diethylamino,ethylmethylamino, diisopropylamino), an aryl (e.g., phenyl, naphthyl,anthranyl), a heteroaryl (e.g., a thienyl (2-thienyl, 3-thienyl), afuryl (2-furyl, 3-furyl), a pyrrolyl (2-pyrrolyl, 3-pyrrolyl), a pyridyl(2-pyridyl, 3-pyridyl, 4-pyridyl), imidazoyl, pyrazoyl, oxazoyl,thiazoyl, isoxazoyl, pyrimidyl, pyrazyl, pyridazoyl), etc. Specifically,the “substituted or unsubstituted heteroaryl” is a thienyl (2-thienyl,3-thienyl), a benzothienyl (e.g., benzo[b]thiophen-2-yl,benzo[b]thiophen-3-yl), a thienothienyl (e.g., thieno[2,3-b]thienyl,thieno[3,2-b]thienyl, thieno[3,2-b:2,3-d]thienyl), a bithiophenyl (e.g.,2,2-bithiophen-5-yl), benzofuranyl, indoyl, indazoyl, benzimidazoyl,benzoxazoyl, benzisoxazoyl, quinolinyl, isoquinolinyl, quinoxalinyl,quinazolinyl, acridinyl, etc.

In some other embodiments of the present invention, coelenteramideanalogue modified at the C-2-position includes the following compounds.

wherein R^(2″) is a group selected from the groups shown below:

and R^(3″) is hydrogen atom, bromine atom, a substituted orunsubstituted aryl, a substituted or unsubstituted arylalkyl, asubstituted or unsubstituted arylalkenyl, a substituted or unsubstitutedarylalkynyl, an alkyl which may optionally be substituted with analicyclic group, an alkenyl which may optionally be substituted with analicyclic group, an alkynyl which may optionally be substituted with analicyclic group, an alicyclic group, or a heterocyclic group.

As used herein, the “substituted or unsubstituted aryl,” “substituted orunsubstituted arylalkyl,” “substituted or unsubstituted arylalkenyl,”“substituted or unsubstituted arylalkynyl,” “alkyl which may optionallybe substituted with an alicyclic group,” “an alkenyl which mayoptionally be substituted with an alicyclic group,” “alkynyl which mayoptionally be substituted with an alicyclic group,” “alicyclic group”and “heterocyclic group” shown by R^(3′″) are the same as those definedfor R^(3″).

In a preferred embodiment of the present invention, coelenteramideanalogue includes the following compounds.

The fluorescent protein containing coelenteramide analogue in someembodiments of the present invention has the maximum fluorescencewavelength of 485 nm or more, for example, 485 nm or more, 486 nm ormore, 487 nm or more, 488 nm or more, 489 nm or more, 490 nm or more,491 nm or more, 492 nm or more, 493 nm or more, 494 nm or more, 495 nmor more, 496 nm or more, 497 nm or more, 498 nm or more, 499 nm or more,500 nm or more, 510 nm or more, 515 nm or more, 520 nm or more, 525 nmor more, 530 nm or more, 535 nm or more, 540 nm or more, and 545 nm ormore. More specifically, in coelenteramide analogues (4a to 4z)described above, coelenteramide analogue which can shift the maximumfluorescence wavelength to 485 nm or more includes 4a to 4l, 4n to 4pand 4u to 4z, preferably, 4a to 4d, 4f to 4l, 4p, 4n, 4v and 4x to 4z,more preferably, 4a, 4b, 4d, 4f to 4i, 4k, 4l and 4p, much morepreferably, 4b, 4f, 4h, 4k and 4p, and most preferably, 4b, 4h and 4p.

The fluorescent protein containing coelenteramide analogue in someembodiments of the present invention shows a stronger fluorescenceintensity than that of the corresponding coelenteramide analogue alone,for example, stronger by 1.5 times or more, 1.6 times or more, 1.7 timesor more, 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more,2.5 times or more, 3.0 times or more, 4.0 times or more, 5.0 times ormore, 10 times or more, 15 times or more, 20 times or more, 30 times ormore, 40 times or more, 50 times or more, 60 times or more, 70 times ormore, 80 times or more, 90 times or more, 100 times or more, 110 timesor more, 120 times or more and 130 times or more. More specifically,among coelenteramide analogues (4a to 4z) described above,coelenteramide analogue in the form of fluorescent protein which canprovide a stronger fluorescence intensity than that of the correspondingcoelenteramide analogue alone includes 4a to 4p and 4r to 4z,preferably, 4a to 4g, 4i to 4p and 4r to 4z, more preferably, 4a, 4f, 4gand 4w, and most preferably, 4a, 4f and 4w.

The fluorescent protein containing coelenteramide analogue in someembodiments of the present invention provides different fluorescencespectra from those of the corresponding coelenteramide analogue alone.The maximum fluorescence wavelengths in the two cases are different by±5 nm or more, ±10 nm or more, ±15 nm or more, ±20 nm or more, ±25 nm ormore, ±30 nm or more, ±35 nm or more, ±40 nm or more, ±45 nm or more,±50 nm or more, ±55 nm or more, ±60 nm or more, ±65 nm or more, ±70 nmor more, ±75 nm or more, ±80 nm or more, ±85 nm or more, ±90 nm or more,±95 nm or more, or ±100 nm or more. More specifically, coelenteramideanalogue which can change the maximum fluorescence intensity between thefluorescent protein and the corresponding coelenteramide alone includes4a to 4n, 4p, 4r, 4s and 4u to 4z, preferably, 4a to 4e, 4f, 4h, 4i, 4k,4m, 4n, 4p, 4u and 4w to 4z, among coelenteramide analogues (4a to 4z)described above.

4. Method for Producing Coelenteramide Analogue of the Present Invention4.1. Coelenteramide Analogue Modified at the C-3 Position

In coelenteramide analogue of the present invention, coelenteramideanalogue modified at the C-3 position represented by general formula(II) below:

(wherein R^(3′) is the same as defined above) can be produced asfollows.

That is, coelenteramide analogue represented by general formula (II) canbe produced by reacting a compound represented by general formula (II-1)shown below:

(wherein R^(3′) is the same as defined above and Y^(1′) is hydrogen atomor a protecting group) with a compound represented by general formula(II-2) shown below:

(wherein X is a splitting-off group and Y^(2′) is hydrogen atom or aprotecting group) to give a compound represented by general formula(II-3) shown below:

(wherein R^(3′), Y^(1′) and Y^(2′) are the same as defined above), orthe like.

As used herein, the protecting groups shown by Y^(1′) and Y^(2′)independently represent, e.g., tert-butyldimethylsilyl (TBDMS),tert-butyldiphenylsilyl (TBDPS), trimethylsilyl (TMS), triethylsilyl(TES), triisopropylsilyl (TIPS), methoxymethyl (MOM),2-methoxethoxyymethyl (MEM), 2-tetrahydropyranyl (THP), methyl (Me),tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), acetyl (Ac) orbenzoyl (Bz); particularly preferred is TBDMS, Me, Bn or Ac.

The splitting-off group shown by X includes, for example, a halogen(e.g., chlorine, fluorine, bromine or iodine), a reactive residue of asulfonic acid (e.g., methanesulfonyloxy, benzenesulfonyloxy ortoluenesulfonyloxy), and an acyloxy for forming an acid anhydride (e.g.,(4-Y^(2′)O)C₆H₄CH₂COO— (wherein Y^(2′) is the same as defined above)).Among them, a halogen is preferred and chlorine is more preferred.

The compound represented by general formula (II-1) can be produced byknown production methods, for example, the methods described in Kishi etal. (1972) Tetrahedron Lett. 13, 2747-2748, Adamczyk et al. (2001) Org.Prep. Proced. Int. 33, 477-485 or Wael, F. D. et al. (2009) Bioorg. Med.Chem. 17, 4336-4344, or modifications of these methods.

The compound represented by general formula (II-2) can be produced byknown production methods. Specifically, any compound can be producedeither by reacting the corresponding carboxylic acid with an excess ofthionyl chloride while heating to reflux followed by concentration underreduced pressure, or by reacting the corresponding carboxylic acid withoxalyl dichloride in a dichloromethane solvent in the presence of acatalytic amount of N,N-dimethylformamide (DMF) followed byconcentration under reduced pressure. Alternatively, the compound mayalso be commercially available.

The compound represented by general formula (II-3) can be produced byknown production methods. Specifically, any compound can be produced byreacting the compound represented by general formula (II-1) with thecompound represented by general formula (II-2), e.g., in an organicsolvent in the presence of a base or in a basic organic solvent.

In the compound represented by general formula (II-3) thus producedwherein Y^(1′) and Y^(2′) represent protecting groups, coelenteramideanalogue represented by general formula (II) can be produced by removingthe protecting groups from the compound represented by general formula(II-3). More specifically, coelenteramide analogue represented bygeneral formula (II) can be produced by the method described in EXAMPLESbelow or modifications thereof.

Herein, where the protecting groups (Y^(1′) and Y^(2′)) are, e.g.,TBDMS, the reagents used in the method for producing coelenteramideanalogue represented by general formula (II) are fluorine reagents suchas tetrabutylammonium fluoride (TBAF), potassium fluoride, hydrofluoricacid, etc. or acids such as acetic acid, hydrochloric acid, hydrobromicacid, hydroiodic acid, hydrofluoric acid, perchloric acid, sulfuricacid, phosphoric acid, nitric acid, etc. These reagents may be usedalone or as a mixture thereof. TBAF is particularly preferred.

Where the protecting groups (Y^(1′) and Y^(2′)) are, e.g., TBDMS,various solvents can be used as the solvent in the method for producingcoelenteramide analogue represented by general formula (II), andexamples include tetrahydrofuran (THF), dioxane, ether, acetonitrile,ethyl acetate, acetone, N,N-dimethylformamide (DMF), dimethylsulfoxide(DMSO), toluene, dichloromethane, methanol, ethanol, butanol, water,etc. These solvents can be used alone or as a mixture thereof.

In the method for producing coelenteramide analogue represented bygeneral formula (II) where the protecting groups (Y^(1′) and Y²) are,e.g., TBDMS, the reaction temperature and reaction time are notparticularly limited but are generally at −20° C. to 200° C. for 10minutes to 24 hours, preferably at 0° C. to 100° C. for 10 minutes to 6hours, more preferably, at room temperature to 50° C. for 30 minutes to2 hours.

Further when the protecting groups (Y^(1′) and Y^(2′)) are, e.g., Me,various reagents can be used as the reagents in the method for producingcoelenteramide analogue represented by general formula (II) inaccordance with the method described in Inouye & Hosoya (2009) Biochem.Biophys. Res. Commun. 386, 617-622 or modifications thereof. Forexample, boron tribromide or pyridinium chloride can be used, and borontribromide is particularly preferred.

In the method for producing coelenteramide analogue represented bygeneral formula (II), solvents can be used. When the protecting groups(Y^(1′) and Y^(2′)) are, e.g., Me, various solvents can be used andexamples are dichloromethane, chloroform, tetrahydrofuran (THF),dioxane, ether, acetonitrile, ethyl acetate, acetone,N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), toluene, etc.These solvents can be used alone or as a mixture thereof.

In the method for producing coelenteramide analogues represented bygeneral formula (II), where the protecting groups (Y^(1′) and Y^(2′))are, e.g., Me, the reaction temperature and reaction time are notparticularly limited but are generally at −78° C. to 200° C. for 10minutes to 72 hours, preferably, 0° C. to 150° C. for 30 minutes to 36hours, and more preferably, at room temperature to 60° C. for an hour to12 hours.

4.2. Coelenteramide Analogue Modified at the C-2 Position 4.2.1.Coelenteramide Analogue Represented by General Formula (III)

In coelenteramide analogues of the present invention, coelenteramideanalogue modified at the C-2 position, which is represented by generalformula (III) shown below:

(wherein Z¹ and R^(3″) are the same as defined above) can be produced asfollows.

The above compound can be produced by first reacting a compoundrepresented by general formula (III-1) shown below:

(wherein R^(3″) is the same as defined above and Y^(1″) is hydrogen atomor a protecting group) with a compound represented by general formula(III-2) shown below:

(wherein Z¹ is the same as defined above and Y^(2″) is hydrogen atom ora protecting group) to give a compound represented by general formula(III-3) shown below:

(wherein R^(3″), Y^(1″), Y^(2″) and Z¹ are the same as defined above),and so on.

As used herein, the “protecting groups” shown by Y^(1″) and Y^(2″) arethe same as those described for Y^(1′) and Y^(2′).

The compound represented by general formula (III-1) can be produced byknown production methods, for example, the methods described in Y. Kishiet al., Tetrahedron Lett., 13, 2747-2748 (1972), M. Adamczyk et al.,Org. Prep. Proced. Int., 33, 477-485 (2001), or F. D. Wael et al.,Bioorg. Med Chem., 17, 4336-4344 (2009), or modifications of thesemethods.

The compound represented by general formula (III-2) can be produced byknown production methods, for example, the method described in S. Knaggset al., Org. Biomol. Chem., 3, 4002-4010 (2005) or modificationsthereof. Alternatively, the compound is commercially available.

The compound represented by general formula (III-3) can be produced byknown production methods. Specifically, any compound can be produced byreacting the compound represented by general formula (III-1) with thecompound represented by general formula (III-2), e.g., in an organicsolvent in the presence of a base or in a basic organic solvent.

In the compound represented by general formula (III-3) thus producedwherein Y^(1″) and Y^(2″) represent protecting groups, coelenteramideanalogues represented by general formula (III) can be produced byremoving the protecting groups from the compound represented by generalformula (III-3). More specifically, coelenteramide analogues representedby general formula (III) can be produced by the method described inEXAMPLES below or modifications thereof.

Herein, where the protecting groups (Y^(1″) and Y^(2″)) are, e.g.,TBDMS, the reagents used in the method for producing coelenteramideanalogues represented by general formula (III) are fluorine reagentssuch as tetrabutylammonium fluoride (TBAF), potassium fluoride,hydrofluoric acid, etc. or acids such as acetic acid, hydrochloric acid,hydrobromic acid, hydroiodic acid, hydrofluoric acid, perchloric acid,sulfuric acid, phosphoric acid, nitric acid, etc. These reagents may beused alone or as a mixture thereof. TBAF is particularly preferred.

Where the protecting groups (Y^(1″) and Y^(2″)) are, e.g., TBDMS,various solvents can be used as the solvent in the method for producingcoelenteramide analogues represented by general formula (I), andexamples include tetrahydrofuran (THF), dioxane, ether, acetonitrile,ethyl acetate, acetone, N,N-dimethylformamide (DMF), dimethylsulfoxide(DMSO), toluene, dichloromethane, methanol, ethanol, butanol, water,etc. These solvents can be used alone or as a mixture thereof.

In the method for producing coelenteramide analogues represented bygeneral formula (III), when the protecting groups (Y^(1″) and Y^(2″))are, e.g., TBDMS, the reaction temperature and reaction time are notparticularly limited but are generally at −20° C. to 200° C. for 10minutes to 24 hours, preferably, at 0° C. to 100° C. for 10 minutes to 6hours, and more preferably, at room temperature to 50° C. for 30 minutesto 2 hours.

Also, when the protecting groups (Y^(1″) and Y^(2″)) are, e.g., Me,various reagents can be used as the reagents in the method for producingcoelenteramide analogues represented by general formula (III) inaccordance with the method described in Inouye & Hosoya, Biochem.Biophys. Res. Commun., 386, 617-622 (2009), or modifications thereof.For example, boron tribromide or pyridinium chloride can be used, andboron tribromide is particularly preferred.

In the method for producing coelenteramide analogues represented bygeneral formula (III), solvents can be used and where the protectinggroups (Y^(1″) and Y^(2″)) are, e.g., Me, various solvents can be used.Examples are dichloromethane, chloroform, tetrahydrofuran (THF),dioxane, ether, acetonitrile, ethyl acetate, acetone,N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), toluene, etc.These solvents can be used alone or as a mixture thereof.

In the method for producing coelenteramide analogues represented bygeneral formula (III) where the protecting groups (Y^(1″) and Y^(2″))are, e.g., Me, the reaction temperature and reaction time are notparticularly limited but are generally at −78° C. to 200° C. for 10minutes to 72 hours, preferably, at 0° C. to 150° C. for 30 minutes to36 hours, and more preferably, at room temperature to 60° C. for an hourto 12 hours.

4.2.2. Coelenteramide Analogue Represented by General Formula (IV)

In coelenteramide analogues of the present invention, coelenteramideanalogue represented by general formula (IV) shown below, which ismodified at the C-2 position can be prepared as follows:

(wherein R^(2′″) and R^(3′″) are the same as defined above).

That is, a compound represented by general formula (IV-1) shown below:

(wherein R^(3′″) is the same as defined above and Y^(1′″) is hydrogenatom or a protecting group) is reacted with a compound represented bygeneral formula (IV-2) shown below.

(wherein X is a splitting-off group and R^(2″) is a group selected from:

(wherein Y^(1′″) is hydrogen atom or a protecting group) to give acompound represented by general formula (IV-3-1) or (IV-3-2) shownbelow:

(wherein R^(2″), R^(3″) and Y^(1′″) are the same as defined above), andso on.

Herein, the splitting off group shown by X includes, for example, ahalogen (e.g., chlorine, fluorine, bromine or iodine), a reactiveresidue of a sulfonic acid (e.g., methanesulfonyloxy, benzenesulfonyloxyor toluenesulfonyloxy), and an acyloxy for forming an acid anhydride(e.g., (4-Y^(1′″)O)C₆H₄CH₂COO— (wherein Y^(1′″) is the same as definedabove)). Among them, a halogen is preferred and chlorine is morepreferred.

The compound represented by general formula (IV-1) can be produced byknown production methods, for example, the methods described in Kishi etal., Tetrahedron Lett., 13, 2747-2748 (1972), Adamczyk et al., Org.Prep. Proced. Int., 33, 477-485 (2001), or modifications of thesemethods.

The compound represented by general formula (IV-2) can be produced byknown production methods. Specifically, any compound can be producedeither by reacting the corresponding carboxylic acid with an excess ofthionyl chloride while heating to reflux followed by concentration underreduced pressure, or by reacting the corresponding carboxylic acid withoxalyl dichloride in a dichloromethane solvent in the presence of acatalytic amount of N,N-dimethylformamide (DMF) followed byconcentration under reduced pressure. Alternatively, the compound mayalso be commercially available.

The compound represented by general formula (IV-3-1) or general formula(IV-3-2) can be produced by known production methods. Specifically, anycompound can be produced by reacting the compound represented by generalformula (IV-1) with the compound represented by general formula (IV-2),e.g., in an organic solvent in the presence of a base or in a basicorganic solvent.

In the compound represented by general formula (IV-3-1) or generalformula (IV-3-2) thus produced wherein Y^(1′″) and Y^(2′″) representprotecting groups, coelenteramide analogues represented by generalformula (IV) can be produced by removing the protecting groups from thecompound represented by general formula (IV-3-1) or general formula(IV-3-2). More specifically, coelenteramide analogues represented bygeneral formula (IV) can be produced by the method described in EXAMPLESbelow or modifications thereof.

Herein, where the protecting groups (Y^(1′″) and Y^(2′″)) are, e.g.,TBDMS, the reagents used in the method for producing coelenteramideanalogues represented by general formula (IV) are fluorine reagents suchas tetrabutylammonium fluoride (TBAF), potassium fluoride, hydrofluoricacid, etc. or acids such as acetic acid, hydrochloric acid, hydrobromicacid, hydroiodic acid, hydrofluoric acid, perchloric acid, sulfuricacid, phosphoric acid, nitric acid, etc. These reagents may be usedalone or as a mixture thereof. TBAF is particularly preferred.

Where the protecting groups (Y^(1′″) and Y^(2′″)) are, e.g., TBDMS,various solvents can be used as the solvent in the method for producingcoelenteramide analogues represented by general formula (IV), andexamples include tetrahydrofuran (THF), dioxane, ether, acetonitrile,ethyl acetate, acetone, N,N-dimethylformamide (DMF), dimethylsulfoxide(DMSO), toluene, dichloromethane, methanol, ethanol, butanol, water,etc. These solvents can be used alone or as a mixture thereof.

In the method for producing coelenteramide analogue represented bygeneral formula (IV) wherein the protecting groups (Y^(1′″) and Y^(2′″))are, e.g., TBDMS, the reaction temperature and reaction time are notparticularly limited but are generally at −20° C. to 200° C. for 10minutes to 24 hours, preferably, at 0° C. to 100° C. for 10 minutes to 6hours, and more preferably, at room temperature to 50° C. for 30 minutesto 2 hours.

Further when the protecting groups (Y^(1′″) and Y^(2′″)) are, e.g., Me,various reagents can be used as the reagent in the method for producingcoelenteramide analogue represented by general formula (IV) inaccordance with the method described in Inouye & Hosoya (2009) Biochem.Biophys. Res. Commun. 386, 617-622 or modifications thereof. Forexample, boron tribromide or pyridinium chloride can be used, and borontribromide is particularly preferred.

In the method for producing coelenteramide analogue represented bygeneral formula (IV), solvents can be used. When the protecting groups(Y^(1′″) and Y^(2′″)) are, e.g., Me, various solvents can be used andexamples are dichloromethane, chloroform, tetrahydrofuran (THF),dioxane, ether, acetonitrile, ethyl acetate, acetone,N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), toluene, etc.These solvents can be used alone or as a mixture thereof.

In the method for producing coelenteramide analogue represented bygeneral formula (IV) where the protecting groups (Y^(1′″) and Y^(2′″))are, e.g., Me, the reaction temperature and reaction time are notparticularly limited but are generally at −78° C. to 200° C. for 10minutes to 72 hours, preferably, at 0° C. to 150° C. for 30 minutes to36 hours, and more preferably, at room temperature to 60° C. for an hourto 12 hours.

5. Method for Producing the Calcium-Binding Photoprotein

5.1. Production from Coelenterazine Analogue

The calcium-binding photoprotein of the present invention can beproduced or regenerated by contacting coelenterazine analogue of thepresent invention described above with an apoprotein of thecalcium-binding photoprotein thereby to obtain the calcium-bindingphotoprotein.

As used herein, the term “contact” means that coelenterazine analogue ofthe present invention and an apoprotein of the calcium-bindingphotoprotein are allowed to be present in the same reaction system, andincludes, for example, states that an apoprotein of the calcium-bindingphotoprotein is added to a container charged with coelenterazineanalogue of the present invention, coelenterazine analogue of thepresent invention is added to a container charged with an apoprotein ofthe calcium-binding photoprotein, coelenterazine analogue of the presentinvention is mixed with an apoprotein of the calcium-bindingphotoprotein, and the like.

In some embodiments of the present invention, the contact is effected ata low temperature in the presence of a reducing agent (e.g.,mercaptoethanol, dithiothreitol, etc.) and oxygen. More specifically,the calcium-binding photoprotein of the present invention can beproduced or regenerated by the methods described in, e.g., Shimomura, O.et al. Biochem. J. 251, 405-410 (1988), Shimomura, O. et al. Biochem. J.261, 913-920 (1989), etc. The calcium-binding photoprotein of thepresent invention exists as a complex which, in the presence of oxygen,generates from a peroxide of coelenterazine analogue produced fromcoelenterazine analogue of the invention and molecular oxygen, and anapoprotein. When calcium ions bind to the complex described above, thecomplex emits a transient light to generate coelenteramide analogue asthe oxide of coelenterazine analogue and carbon dioxide. The complexdescribed above is sometimes referred to as the “photoprotein of thepresent invention.”

Examples of the apoprotein used to produce the photoprotein of thepresent invention include apoaequorin, apoclytin-I, apoclytin-II,apobelin, apomitrocomin, apomineopsin, apobervoin, and the like. In someembodiments of the present invention, the apoprotein is apoaequorin,apobelin, apoclytin-1, apoclytin-II, mitrocomin, etc., e.g.,apoaequorin. These apoproteins may be obtained from natural sources orgenetically engineered. Furthermore, the amino acid sequence may also bemutated from the native sequence by gene recombination technology, aslong as the apoproteins are capable of producing the calcium-bindingphotoprotein.

The nucleotide sequences and amino acid sequences of the apoproteins ofphotoproteins obtained from the nature (native apoproteins) are asfollows. The nucleotide sequence and amino acid sequence of nativeapoaequorin are shown by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.The nucleotide sequence and amino acid sequence of native apoclytin-Iare shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The nucleotidesequence and amino acid sequence of native apoclytin-II are shown by SEQID NO: 5 and SEQ ID NO: 6, respectively. The nucleotide sequence andamino acid sequence of native apomitrocomin are shown by SEQ ID NO: 7and SEQ ID NO: 8, respectively. The nucleotide sequence and amino acidsequence of native apobelin are shown by SEQ ID NO: 9 and SEQ ID NO: 10,respectively. The nucleotide sequence and amino acid sequence of nativeapobervoin are shown by SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

The apoprotein mutated by recombinant technology is a protein selectedfrom the group consisting of (a) to (c) below:

(a) a protein comprising the amino acid sequence of native apoprotein inwhich 1 or more amino acids are deleted, substituted, inserted and/oradded, and having the activity or function of the apoprotein of thecalcium-binding photoprotein;

(b) a protein comprising an amino acid sequence which has 90% or morehomology to the amino acid sequence of native apoprotein, and having theactivity or function of the apoprotein of the calcium-bindingphotoprotein; and,

(c) a protein comprising an amino acid sequence encoded by apolynucleotide that hybridizes under stringent conditions to apolynucleotide comprising a nucleotide sequence complementary to thenucleotide sequence of native apoprotein, and having the activity orfunction of the apoprotein of the calcium-binding photoprotein.

Examples of the “native apoprotein” described above are apoaequorin,apoclytin-I, apoclytin-II, apobelin, apomitrocomin, apomineopsin,apobervoin, etc. In an embodiment of the present invention, theapoprotein is apoaequorin, apoclytin-I, apoclytin-II, apobelin,apomitrocomin, etc., preferably apoaequorin. The amino acid sequencesand nucleotide sequences of these native apoproteins are the same asdescribed above.

The “activity or function of the apoprotein of the calcium-bindingphotoprotein” means the activity or function of the apoprotein whichbinds to a peroxide of coelenterazine or a peroxide of coelenterazineanalogue to produce the calcium-binding photoprotein. Specifically, “theprotein binds to a peroxide of coelenterazine or a peroxide ofcoelenterazine analogue to produce the calcium-binding photoprotein”means not only (1) that the apoprotein binds to the peroxide ofcoelenterazine or the peroxide of coelenterazine analogue to produce thephotoprotein, but also (2) that the apoprotein is brought in contactwith coelenterazine or its analogues in the presence of oxygen toproduce the photoprotein (complex) containing the apoprotein and theperoxide of coelenterazine or the peroxide of coelenterazine analogue.As used herein, the term “contact” means that the apoprotein andcoelenterazine or its analogue are allowed to be present in the samereaction system, and includes, for example, addition of the apoproteinto a container charged with coelenterazine or its analogue, addition ofcoelenterazine or its analogue to a container charged with theapoprotein, mixing of the apoprotein with coelenterazine or itsanalogue, and the like. “Coelenterazine analogue” refers to a compoundcapable of constituting as the apoprotein the calcium-bindingphotoprotein such as aequorin, etc. as in coelenterazine. Examples ofcoelenterazine or its analogue are, in addition to coelenterazineanalogue of the present invention, coelenterazine, h-coelenterazine,f-coelenterazine, cl-coelenterazine, n-coelenterazine,cp-coelenterazine, ch-coelenterazine, hch-coelenterazine,fch-coelenterazine, e-coelenterazine, ef-coelenterazine,ech-coelenterazine, hcp-coelenterazine, and the like. Thesecoelenterazine and analogues of the present invention can be produced,e.g., by the method described in EXAMPLES below or its modifications.The other coelenterazine and analogues thereof can be produced by themethods described in, e.g., Shimomura et al. (1988) Biochem. J. 251,405-410, Shimomura et al. (1989) Biochem. J. 261, 913-920 and Shimomuraet al. (1990) Biochem. J. 270, 309-312, or modifications thereof.Alternatively, these compounds are commercially available from ChissoCorp., Wako Pure Chemicals, Promega Inc., etc. and such commercialproducts may also be used.

The range of “1 or more” in “the amino acid sequence in which 1 or moreamino acids are deleted, substituted, inserted and/or added” is, forexample, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 toseveral), 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1. In general, the lessthe number of amino acids deleted, substituted, inserted or added, themore preferable. In the deletion, substitution, insertion and additionof the amino acid residues described above, two or more may occurconcurrently. Such regions can be acquired by using site-directedmutagenesis described in Sambrook J. et al., Molecular Cloning. ALaboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press(2001); Ausbel F. M. et al., Current Protocols in Molecular Biology,Supplement 1-38, John Wiley and Sons (1987-1997); Nuc. Acids. Res., 10,6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315(1985); Nuc. Acids. Res., 13, 4431 (1985); Proc. Natl. Acad. Sci. USA,82, 488 (1985); etc.

The range of “90% or more” in the “amino acid sequence which has 90% ormore homology” is, for example, 90% or more, 91% or more, 92% or more,93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% ormore, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or99.9% or more. It is generally preferable for the numerical valueindicating the degree of homology to be higher. The homology betweenamino acid sequences or nucleotide sequences can be determined using asequencing program such as BLAST (see, e.g., Altzchul, S. F. et al., J.Mol. Biol., 215, 403 (1990), etc.) or the like. When BLAST is used, thedefault parameters for the respective programs are used.

The “polynucleotide that hybridizes under stringent conditions”described above refers to a polynucleotide (e.g., DNA) which is obtainedby, for example, colony hybridization, plaque hybridization, Southernhybridization, etc., using as a probe all or part of a polynucleotidecomprising a nucleotide sequence complementary to the nucleotidesequence of native apoprotein or a polynucleotide encoding the aminoacid sequence of native apoprotein. Specific examples include apolynucleotide which can be identified by performing hybridization at65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on whichthe polynucleotide from a colony or plaque is immobilized, then washingthe filter at 65° C. with an SSC (saline-sodium citrate) solution havinga concentration of 0.1 to 2 times (a 1×SSC solution is composed of 150mmol/L of sodium chloride and 15 mmol/L of sodium citrate).

Hybridization may be performed in accordance with modifications of themethods described in textbooks of experiment, e.g., Sambrook, J. et al.:Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press (2001), Ausbel F. M. et al., Current Protocolsin Molecular Biology, Supplement 1-38, John Wiley and Sons (1987-1997),Glover D. M. and Hames B. D., DNA Cloning 1: Core Techniques, Apractical Approach, Second Edition, Oxford University Press (1995), etc,or their modifications.

As used herein, “stringent conditions” may refer to less stringentconditions, moderately stringent conditions and highly stringentconditions. The “less stringent conditions” are, for example, theconditions under 5×SSC, 5×Denhardt's solution, 0.5% (w/v) SDS and 50%(v/v) formamide at 32° C. The “moderately stringent conditions” are, forexample, the conditions under 5×SSC, 5×Denhardt's solution, 0.5% (w/v)SDS and 50% (v/v) formamide at 42° C. The “highly stringent conditions”are, for example, the conditions under 5×SSC, 5×Denhardt's solution,0.5% (w/v) SDS and 50% (v/v) formamide at 50° C. The more stringent theconditions are, the higher the complementarity required for doublestrand formation. Specifically, for example, under these conditions, apolynucleotide (e.g., DNA) of higher homology is expected to be obtainedefficiently at higher temperatures, although multiple factors areinvolved in hybridization stringency, including temperature, probeconcentration, probe length, ionic strength, time and baseconcentration; one skilled in the art may appropriately select thesefactors to realize a similar stringency.

When a commercially available kit is used for the hybridization, forexample, AlkPhos Direct Labeling Reagents (manufactured by AmershamPharmacia) can be used. According to the protocol bound to the kit inthis case, incubation with a labeled probe is performed overnight, themembrane is then washed with a primary wash buffer containing 0.1% (w/v)SDS at 55° C., and finally the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having homology of 60% or more, 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more,92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.3%or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more,to the polynucleotide encoding the amino acid sequence of theapoprotein. The homology of an amino acid sequence or a nucleotidesequence can be determined using the method described hereinabove.

Examples of the recombinant apoprotein which can be used in the presentinvention include recombinant aequorin described in Shimomura, O. andInouye, S. Protein Express. Purif. (1999) 16: 91-95, recombinantclytin-I described in Inouye, S. and Sahara, Y. Protein Express. Purif(2007) 53: 384-389, recombinant clytin-II described in Inouye, S. J.Biochem. (2008) 143: 711-717, and the like.

The calcium-binding photoprotein thus produced may be further purifiedPurification of the calcium-binding photoprotein can be performed in aconventional manner of separation/purification. Theseparation/purification includes, for example, precipitation withammonium sulfate, gel filtration chromatography, ion exchangechromatography, affinity chromatography, reversed phase high performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or in anappropriate combination of these techniques.

The photoprotein in some embodiments of the present invention exhibitsdifferent luminescence properties from those of known photoproteins. Thephotoprotein in a preferred embodiment of the present invention providesa longer half decay time of luminescence, which is a time period inwhich the luminescence becomes half of the maximum luminescenceintensity, when compared to a photoprotein containing coelenterazine asa light emitting substrate. Furthermore, the photoprotein in a morepreferred embodiment of the present invention provides differentluminescence spectra from luminescence spectra of the correspondingfluorescent protein prepared by causing luminescence of the photoproteinin the presence of calcium.

5.2. Production from Green Fluorescent Protein (gFP)-Like Protein

As illustrated in FIG. 8, the calcium-binding photoprotein such asaequorin, etc. can be produced by contacting coelenterazine or itsanalogues with the gFP-like protein of the present invention in thepresence of a chelating agent such as EDTA, etc., for sequesteringcalcium ions or divalent or trivalent ions replaceable for the calciumions thereby to obtain the calcium-binding photoprotein. The term“contact” means that the gFP-like protein of the present invention andcoelenterazine or its analogue are allowed to be present in the samereaction system, and includes, for example, addition of the gFP-likeprotein of the present invention to a container charged withcoelenterazine or its analogue, addition of coelenterazine or itsanalogue of the present invention to a container charged with thegFP-like protein of the present invention, mixing of the gFP-likeprotein of the present invention with coelenterazine or its analogue,and the like.

The gFP-like protein of the present invention used to produce thecalcium-binding photoprotein of the present invention is describedhereinbelow.

Examples of coelenterazine or its analogue used to produce thecalcium-binding photoprotein of the present invention include, inaddition to coelenterazine analogue of the present invention,coelenterazine, h-coelenterazine, f-coelenterazine, cl-coelenterazine,n-coelenterazine, cp-coelenterazine, ch-coelenterazine,hch-coelenterazine, fch-coelenterazine, e-coelenterazine,ef-coelenterazine, ech-coelenterazine, hcp-coelenterazine, and the like,preferably coelenterazine, h-coelenterazine and e-coelenterazine. Thesecoelenterazine and analogues thereof are available as described above.

The amount of coelenterazine or its analogue used to produce thecalcium-binding photoprotein is not particularly limited, and is, e.g.,1.2 mol or more per mol of the gFP-like protein.

In the method for producing the calcium-binding photoprotein, thereaction temperature and reaction time are not particularly limited andare, for example, at 0° C. to 42° C. for 0.1 to 2 hours, at 4° C. to 37°C. for 0.1 to 2 hours, or at 4° C. to 15° C. for 0.1 to 24 hours.

It is preferred to carry out the reaction of the gFP-like protein of thepresent invention with coelenterazine or its analogue in the presence ofthe chelating agent for sequestering calcium ions or divalent ortrivalent ions replaceable for the calcium ions. The chelating agentused to produce the gFP-like protein in the present invention is thesame as described above, and may be any agent but is not particularlylimited, so long as it strongly binds to calcium ions or divalent ortrivalent ions replaceable for the calcium ions. Examples of thechelating agent include ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CyDTA),N-(2-hydroxyethyl)iminodiacetic acid (HIDA) and the like. As usedherein, the calcium ions or divalent or trivalent ions replaceable forthe calcium ions are the same as those described above.

The amount of the chelating agent used to produce the gFP-like proteinis not particularly limited unless its concentration affectsregeneration of the gFP-like protein. Since it is demonstrated that 3mols of calcium ions bind to 1 mol of ion apoaequorin, e.g., 3 mols ormore are preferred.

In a more preferred embodiment of the present invention, the fluorescentprotein is reacted with coelenterazine or its analogue in the presenceof a reducing agent. Examples of the reducing agent used herein includedithiothreitol (DTT), mercaptoethanol, etc. The amount of the reducingagent used to produce the calcium-binding photoprotein is notparticularly limited, so long as the amount does not affect theregeneration, but the concentration is preferably sufficient to preventthe S—S bonds formed by the presence of cysteine residues at the threepositions of apoaequorin. The concentration is, for example, 1 mMdithiothreitol or 0.1% (v/v) mercaptoethanol in a final concentration.

6. Use of the Photoprotein of the Invention (1) Detection orQuantitative Determination of Calcium Ions

The photoprotein of the present invention obtained as described above isa non-covalent complex of an apoprotein and a peroxide of coelenterazineanalogue formed from coelenterazine analogue and molecular oxygen and isa photoprotein (holoprotein) that emits light by the action of calciumions. Therefore, the photoprotein of the invention can be used for thedetection or quantitative determination of calcium ions.

The detection or quantitative determination of calcium ions can beperformed, for example, by adding a sample solution directly to asolution of the photoprotein and measuring the luminescence generated.Alternatively, calcium ions can also be detected or quantified by addinga solution of the photoprotein to a sample solution and measuring theluminescence generated. Also, the photoprotein above may be formed bypreviously contacting an aqueous apoprotein solution with coelenterazineanalogue of the present invention prior to its addition to the assaysystem for the detection or quantitative determination of calcium ionsand the resulting photoprotein may be provided for use. The photoproteincomprising an apoprotein and a peroxide of coelenterazine analogue mayalso be formed in the assay system by contacting the apoprotein withcoelenterazine analogue. The photoprotein formed is a complex(photoprotein) of the apoprotein and the peroxide of coelenterazineanalogue of the invention. The complex (i.e., the photoprotein of thepresent invention) emits light dependently on the calcium ionconcentration.

The detection or quantitative determination of calcium ions can beperformed by measuring the luminescence of the photoprotein of theinvention through the action of calcium ions, using a luminometer.Luminometers which may be used include commercially availableinstruments, such as a Centro LB 960 (manufactured by Berthold, Inc.),etc. The calcium ion concentration can be quantitatively determined bypreparing a luminescence standard curve for known calcium ionconcentrations using the photoprotein.

Coelenterazine analogue of the present invention may also be used forthe detection of changes in the intracellular calcium ion concentrationunder the physiological conditions by preparing the photoproteincomprising an apoprotein and a peroxide of coelenterazine analogue andinjecting the photoprotein directly into cells by means ofmicroinjection, etc.

Coelenterazine analogue of the present invention may also be used toproduce the photoprotein, which is performed, in addition to theinjection using techniques such as microinjection, by intracellularlyexpressing a gene for the apoprotein (a polynucleotide encoding theapoprotein) to produce the protein in the cells and addingcoelenterazine analogue of the present invention to the resultingapoprotein from the external cells.

Using the photoprotein of the present invention thus introduced intocells or produced in cells, changes in the intracellular calcium ionconcentration caused by external stimulation (e.g., stimulation withreceptor-associated drugs, etc.) can also be determined.

(2) Use as a Reporter Protein, Etc. Utilizing Luminescence

The photoprotein of the present invention can also be used as a reporterprotein to determine the transcription activity of a promoter, etc. Apolynucleotide encoding an apoprotein is fused to a target promoter orother expression control sequence (e.g., an enhancer, etc.) to constructa vector. The vector described above is transfected to a host cell, andcoelenterazine analogue of the present invention is brought in contactwith the host cell. By detecting the luminescence from the photoproteinof the present invention, the activity of the target promoter or otherexpression control sequence can be determined. As used herein, the term“contact” means that a host cell and coelenterazine analogue of thepresent invention are allowed to be present in the same culture/reactionsystem, and includes, for example, addition of coelenterazine analogueof the present invention to a culture container charged with a hostcell, mixing of a host cell with coelenterazine analogue of the presentinvention, culture of a host cell in the presence of coelenterazineanalogue of the present invention, and the like.

The present invention further provides a kit used for the measurement oftranscription activity of a promoter, etc. In some embodiments of thepresent invention, the kit comprises coelenterazine analogue of thepresent invention, cells containing a polynucleotide (e.g., DNA)encoding the apoprotein of the calcium-binding photoprotein, etc.Reagent such as coelenterazine analogue of the present invention, etc.may be dissolved in a suitable solvent and prepared to be suitable forstorage. The solvent which may be used is at least one selected from thegroup consisting of water, ethanol, various buffer solutions, and thelike. The kit may additionally comprise, if necessary, at least oneselected from the group consisting of a container designed therefor,other necessary accessories and an instruction manual, and the like.

(3) Use as a Detection Marker Utilizing Luminescence

The photoprotein of the present invention can be used as a marker fordetection by luminescence. The detection marker of the present inventioncan be used to detect a target substance in, e.g., immunoassay,hybridization assay, etc. The photoprotein of the present invention canbe used in the form bound to a target substance (protein, nucleic acid,etc.) in a conventional manner including chemical modification.Detection methods using the detection marker can be performed in aconventional manner. The detection marker of the present invention canalso be used to determine the distribution of a target substance byexpressing the marker, e.g., as a fusion protein of the apoprotein andthe target substance, then inserting the fusion protein into cells bymeans of microinjection or the like and contacting them withcoelenterazine analogue of the present invention thereby to produce thephotoprotein of the present invention. As used herein, the term“contact” means that cells and coelenterazine analogue of the presentinvention are allowed to be present in the same culture/reaction system,and includes, for example, addition of coelenterazine analogue of thepresent invention to a culture container charged with cells, mixing ofcells with coelenterazine analogue of the present invention, culture ofhost cells in the presence of coelenterazine analogue of the presentinvention, and the like.

The distribution of such a target protein, etc. can be determined by amethod for detection such as luminescence imaging. The apoprotein canalso be used after expression in cells, in addition to the insertioninto cells by means of microinjection, etc.

The present invention further provides a kit used for the detection of atarget substance in an immunoassay, hybridization assay, etc. The kit insome embodiments of the present invention comprises the photoprotein ofthe present invention. The kit in another embodiment of the presentinvention comprises coelenterazine analogue of the present invention, acell containing a polynucleotide (e.g., DNA) encoding the apoprotein ofthe calcium-binding photoprotein, etc. Reagent such as coelenterazineanalogue, etc. may be dissolved in a suitable solvent and prepared to besuitable for storage. The solvent which may be used is at least oneselected from the group consisting of water, ethanol, various buffersolutions, and the like. The kit may additionally comprise, ifnecessary, at least one selected from the group consisting of acontainer designed therefor, other necessary accessories and aninstruction manual, and the like.

(4) Material for Amusement Supplies

The complex (photoprotein of the present invention) comprising theapoprotein and a peroxide of coelenterazine analogue of the presentinvention emits light only by binding to a trace of calcium ions. Thephotoprotein of the present invention can be preferably used as aluminescence material for amusement supplies. Examples of such amusementsupplies are luminescent soap bubbles, luminescent ice, luminescentcandies, luminescent color paints, etc. The amusement supplies of thepresent invention can be prepared in a conventional manner.

(5) Bioluminescence Resonance Energy Transfer (BRET) Method

The photoprotein of the present invention can be used for analysesincluding an analysis of biological functions, an assay for enzymeactivities, etc., based on the principle of intermolecular interactionby the bioluminescence resonance energy transfer (BRET) method.

For example, using the photoprotein of the invention as a donor proteinand an organic compound or a fluorescent protein as an acceptor protein,the interaction between the proteins can be detected by generatingbioluminescence resonance energy transfer (BRET) between them. In someembodiments of the present invention, the organic compound used as anacceptor protein is Hoechist 3342, Indo-1, DAP1, etc. In anotherembodiment of the present invention, the fluorescent protein used as anacceptor protein is a green fluorescent protein (GFP), a bluefluorescent protein (BFP), a mutant GFP fluorescent protein, phycobilin,etc. In a preferred embodiment of the present invention, thephysiological functions to be analyzed include an orphan receptor (inparticular, G-protein conjugated receptor), apoptosis, transcriptionregulation by gene expression, etc. Still in a preferred embodiment ofthe present invention, the enzyme to be analyzed is protease, esterase,kinase, etc.

Analysis of the physiological functions by the BRET method may beperformed by known methods, for example, by modifications of the methodsdescribed in Biochem. J. 2005, 385, 625-637, Expert Opin. Ther. Targets,2007 11: 541-556, etc. Assay for the enzyme activity may also beperformed by known methods, for example, by modifications of the methodsdescribed in Nat. Methods 2006, 3.165-174, Biotechnol. J. 2008, 3:311-324, etc.

The present invention further provides a kit used for the analysismethod described above. The kit comprises the photoprotein of thepresent invention and the organic compound and/or the fluorescentprotein. Reagent such as the photoprotein of the present invention,organic compounds, fluorescent proteins, etc. may be dissolved in asuitable solvent and prepared to be suitable for storage. The solventwhich may be used is at least one selected from the group consisting ofwater, ethanol, various buffer solutions, and the like. The kit mayadditionally comprise, if necessary, at least one selected from thegroup consisting of a container designed therefor, other necessaryaccessories and an instruction manual, and the like.

7. Fluorescent Protein

As illustrated in FIG. 8, the blue fluorescent protein (BFP)-likefluorescent protein can be produced by contacting coelenteramide or itsanalogues with an apoprotein such as apoaequorin, etc. Alternatively,the BFP-like fluorescent protein can also be produced by generatingluminescence through contact of the calcium-binding photoprotein such asaequorin, etc. with calcium ions or bivalent or trivalent ionsreplaceable for the calcium ions to obtain the BFP-like protein.

On the other hand, the green fluorescent protein (gFP)-like fluorescentprotein can be produced by contacting coelenteramide or its analogueswith an apoprotein such as apoaequorin, etc. in the presence of achelating agent for sequestering calcium ions or divalent or trivalentions replaceable for the calcium ions thereby to obtain the gFP-likefluorescent protein. Alternatively, the gFP-like fluorescent protein canalso be produced by treating the BFP-like fluorescent protein with achelating agent such as EDTA, etc., for sequestering calcium ions ordivalent or trivalent ions replaceable for the calcium ions.

7.1. Blue Fluorescent Protein (BFP)-Like Fluorescent Protein 7.1.1.Production of Blue Fluorescent Protein (BFP)-Like Fluorescent Protein

The blue fluorescent protein (BFP)-like fluorescent protein of thepresent invention is a complex of the calcium-binding photoprotein andcoelenteramide analogue of the present invention which is coordinated tothe apoprotein of the photoprotein. That is, the BFP-like fluorescentprotein of the present invention comprises coelenteramide analogue ofthe present invention, the apoprotein of the calcium-bindingphotoprotein and calcium ions or divalent or trivalent ions replaceablefor the calcium ions. The BFP-like fluorescent protein can emitfluorescence upon excitation of light and can also generate luminescenceupon contact of the BFP-like fluorescent protein with coelenterazine orits analogue.

According to the present invention, the BFP-like fluorescent protein isprepared from coelenteramide analogue of the present invention asfollows. That is, coelenteramide analogue of the present invention isbrought in contact with the apoprotein of the calcium-bindingphotoprotein in the presence of calcium ions or divalent or trivalentions replaceable for the calcium ions thereby to obtain the BFP-likefluorescent protein. As used herein, the term “contact” means that theapoprotein and coelenteramide analogue are allowed to be present in thesame reaction system, and includes, for example, addition of theapoprotein to a container charged with coelenteramide analogue, additionof coelenteramide analogue to a container charged with the apoprotein,mixing of the apoprotein with coelenteramide analogue, and the like.

In the present invention, coelenteramide analogue of the presentinvention used to produce the BFP-like fluorescent protein is the sameas described above Examples of coelenteramide analogue of the presentinvention include compounds prepared in EXAMPLES below or by methodsmodified therefrom.

The divalent or trivalent ions replaceable for the calcium ions whichare used in the present invention to produce the BFP-like fluorescentprotein are divalent or trivalent ions which trigger the luminescencereaction when they react with the calcium-binding photoprotein, in placeof calcium ions. In other words, they refer to ions that exert thefunction similar to calcium ions on the calcium-binding photoprotein.Examples of the calcium ions or divalent or trivalent ions replaceablefor the calcium ions include calcium ions (Ca²⁺), magnesium ions (Mg²⁺),strontium ions (Sr²⁺), barium ions (Ba²⁺), lead ions (Pb²⁺), cobalt ions(Co²⁺), nickel ions (Ni²⁺), cadmium ions (Cd²⁺), yttrium ions (Y³⁺),lanthanum ions (La³⁺), samarium ions (Sm³⁺), europium ions (Eu³⁺),dysprosium ions (Dy³⁺), thulium ions (Tm³⁺), ytterbium ions (Yb³⁺), andthe like. Among these ions, the divalent metal ions are preferred, morepreferably the divalent metal ions other than transition metals, e.g.,Ca²⁺, Sr²⁺, Pb²⁺, etc.

In the present invention, the apoprotein of the calcium-bindingphotoprotein used to produce the BFP-like fluorescent protein is thesame as described above.

The amount of coelenteramide analogue of the present invention used toproduce the BFP-like fluorescent protein is not particularly limited andis in a range of, e.g., 1 mol to 5 mol, preferably 1 mol to 2 mol, morepreferably 1 mol to 1.2 mol, based on 1 mol of apoprotein.

In the production of the BFP-like fluorescent protein, preferablycoelenteramide analogue of the present invention is reacted with theapoprotein and calcium ions or divalent or trivalent ions replaceablefor the calcium ions in the presence of a reducing agent. Examples ofthe reducing agent used herein include dithiothreitol (DTT),mercaptoethanol, etc. The amount of the reducing agent used to producethe BFP-like fluorescent protein is not particularly limited so long asthe amount does not affect the regeneration of the BFP-like fluorescentprotein, and the concentration is preferably sufficient to prevent theS—S bonds formed by the presence of cysteine residues at the threepositions of apoaequorin. Such a concentration is, for example, 1 mMdithiothreitol or 0.1% (v/v) mercaptoethanol in a final concentration.

In the method for producing the BFP-like fluorescent protein, thereaction temperature and reaction time are not particularly limited butare generally at 0° C. to 42° C. for 0.1 to 2 hours, at 4° C. to 37° C.for 0.1 to 2 hours, or at 4° C. to 15° C. for 0.1 to 24 hours.

The BFP-like fluorescent protein thus produced may be further purified.Purification of the BFP-like fluorescent protein can be performed in aconventional manner of separation/purification. Theseparation/purification includes, for example, precipitation withammonium sulfate, gel filtration chromatography, ion exchangechromatography, affinity chromatography, reversed phase high performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or in anappropriate combination of these techniques.

7.1.2. Use of Blue Fluorescent Protein (BFP)-Like Fluorescent Protein(1) Use as a Luminescent Catalyst

The BFP-like fluorescent protein of the present invention acts on aluminescence substrate to emit light from the substrate and can be usedas a luminescent catalyst. Therefore, the present invention provides amethod for emitting light, which comprises contacting the BFP-likefluorescent protein of the present invention with coelenterazine or itsanalogues. As used herein, the term “contact” means that the BFP-likefluorescent protein and coelenterazine or its analogue are allowed to bepresent in the same reaction system, and includes, for example, additionof the BFP-like fluorescent protein to a container charged withcoelenterazine or its analogue, addition of coelenterazine or itsanalogue to a container charged with the BFP-like fluorescent protein,mixing of the BFP-like fluorescent protein with coelenterazine or itsanalogue, and the like.

The luminescent substrate used in the method for light emissionaccording to the present invention is, for example, coelenterazine orits analogue. The analogue of coelenterazine includes the same asdescribed above.

These coelenterazine and analogues thereof are brought in contact withthe BFP-like fluorescent protein. By the catalytic action of theBFP-like fluorescent protein contacted, coelenterazine or its analogueis oxidized to the corresponding coelenteramide or its analogue, wherebyluminescence generates (at this time carbon dioxide is released). Theluminescence time is generally 0.5 to 3 hours. However, the luminescencetime can be further prolonged or more shortened, depending uponconditions chosen.

(2) Use as a Reporter Protein

The BFP-like fluorescent protein of the present invention can also beused as a reporter protein to determine the transcription activity of apromoter, etc. A polynucleotide encoding the apoprotein is fused to atarget promoter or other expression control sequence (e.g., an enhancer,etc.) to construct a vector. The vector described above is transfectedto a host cell. Coelenteramide analogue of the present invention andcalcium ions or divalent or trivalent ions replaceable for the calciumions are brought in contact with the host cell. By detecting thefluorescence from the fluorescent protein of the present invention, theactivity of the target promoter or other expression control sequence canbe determined. As used herein, the term “contact” means that a host celland coelenteramide analogue as well as the calcium ions or divalent ortrivalent ions replaceable for the calcium ions are allowed to bepresent in the same culture/reaction system, and includes, for example,addition of coelenteramide analogue and calcium ions or divalent ortrivalent ions replaceable for the calcium ions to a culture containercharged with a host cell, mixing of a host cell with coelenteramideanalogue and calcium ions or divalent or trivalent ions replaceable forthe calcium ions, culture of a host cell in the presence ofcoelenteramide analogue and calcium ions or divalent or trivalent ionsreplaceable for the calcium ions, and the like.

(3) Use as a Detection Marker

The BFP-like fluorescent protein of the present invention can be used asa marker for detection by fluorescence. The detection marker of thepresent invention can be used to detect a target substance in, e.g.,immunoassay, hybridization assay, etc. The BFP-like fluorescent proteinof the present invention can be used in the form bound to a targetsubstance (protein, nucleic acid, etc.) in a conventional mannerincluding chemical modification. Detection methods using the detectionmarker can be performed in a conventional manner.

The detection marker of the present invention can also be used todetermine the distribution of a target substance by expressing themarker, e.g., as a fusion protein of the apoprotein and the targetsubstance, then inserting the fusion protein into cells by means ofmicroinjection or the like, and contacting them with coelenteramideanalogue of the present invention and calcium ions or divalent ortrivalent ions replaceable for the calcium ions. As used herein, theterm “contact” means that cells and coelenteramide analogue as well ascalcium ions or divalent or trivalent ions replaceable for the calciumions are allowed to be present in the same culture/reaction system, andincludes, for example, addition of coelenteramide analogue and calciumions or divalent or trivalent ions replaceable for the calcium ions to aculture container charged with cells, mixing of cells withcoelenteramide analogue and calcium ions or divalent or trivalent ionsreplaceable for the calcium ions, culture of a host cell in the presenceof coelenteramide analogue and calcium ions or divalent or trivalentions replaceable for the calcium ions, and the like.

The distribution of such a target protein, etc. can be determined by amethod for detection such as fluorescence imaging. The apoprotein canalso be used after expression in cells, in addition to the insertioninto cells by means of microinjection, etc.

(4) Material for Amusement Supplies

When excited with light, the BFP-like fluorescent protein of theinvention emits fluorescence. Therefore, the BFP-like fluorescentprotein of the present invention can be preferably used as afluorescence material for amusement supplies. Examples of such amusementsupplies are fluorescent soap bubbles, fluorescent ice, fluorescentcandies, fluorescent color paints, etc. The amusement supplies of theinvention can be prepared in a conventional manner.

(5) Fluorescence Resonance Energy Transfer (FRET) Method

The BFP-like fluorescent protein of the present invention can be usedfor analyses including an analysis of biological functions, an assay (adetermination) for enzyme activities, etc., based on the principle ofintermolecular interaction by the fluorescence resonance energy transfer(FRET) method.

For example, using the BFP-like fluorescent protein of the invention asa donor or an acceptor and an organic compound or another fluorescentprotein as an acceptor or a donor, the interaction between the proteinscan be detected by causing fluorescence resonance energy transfer (FRET)between them. In some embodiments of the present invention, the organiccompound used as an acceptor or a donor is Hoechist 3342, Indo-1, DAP1,etc. In some other embodiments of the present invention, anotherfluorescent protein used as an acceptor or a donor is another greenfluorescent protein (GFP), another blue fluorescent protein (BFP),another mutant GFP fluorescent protein, phycobilin, etc. In a preferredembodiment of the present invention, the physiological functions to beanalyzed include an orphan receptor (in particular, G-protein conjugatedreceptor), apoptosis, transcription regulation by gene expression, etc.In a preferred embodiment of the present invention, the enzyme to beanalyzed is protease, esterase, kinase, etc.

Analysis of the physiological functions by the FRET method may beperformed by known methods, for example, by modifications of the methodsdescribed in Hoffmann, C. et al Nat Methods (2005) 2: 171-176, Paulsson,J. F. et al. Exp. Diabetes Res. 2008: 2008, 865850, etc. Assay for theenzyme activity may also be performed by known methods, for example, bymodifications of the methods described in Ting, A. Y. et al (2001) Proc.Natl. Acad Sci. USA 98: 15003-15008, Evellin, S. et al (2004) Methods.Mol. Biol. 284: 259-270, Palmer A. E. & Tsien, R. Y. (2006) 1:1057-1065,etc.

Still, the present invention provides a kit used for the analysis methoddescribed above. The kit comprises the BFP-like fluorescent protein ofthe present invention and the organic compound and/or the fluorescentprotein. Reagent such as the BFP-like fluorescent protein of the presentinvention, organic compounds, other fluorescent proteins, etc. may bedissolved in a suitable solvent and prepared to be suitable for storage.The solvent which may be used is at least one selected from the groupconsisting of water, ethanol, various buffer solutions, and the like.The kit may additionally comprise, if necessary, at least one selectedfrom the group consisting of a container designed therefor, othernecessary accessories and an instruction manual, and the like.

7.2. Green Fluorescent Protein (gFP)-Like Protein7.2.1. Production of Green Fluorescent Protein (gFP)-Like Protein

The green fluorescent protein (gFP)-like protein of the presentinvention is a complex of the calcium-binding photoprotein andcoelenteramide analogue of the present invention which is coordinated tothe apoprotein of the photoprotein. That is, the gFP-like protein of thepresent invention comprises coelenteramide analogue of the presentinvention and the apoprotein of the calcium-binding photoprotein. On theother hand, the gFP-like protein of the present invention does notcomprise calcium ions or divalent or trivalent ions replaceable for thecalcium ions. The gFP-like protein can emit fluorescence upon excitationof light.

The gFP-like protein of the present invention can be produced bycontacting coelenteramide analogue of the present invention with theapoprotein of the calcium-binding photoprotein in the presence of achelating agent for removing calcium ions or divalent or trivalent ionsreplaceable for the calcium ions thereby to obtain the gFP-like protein.

Alternatively, the gFP-like protein can also be produced by removingcalcium ions or divalent or trivalent ions replaceable for the calciumions from the BFP-like fluorescent protein above to obtain the gFP-likeprotein. The calcium ions or divalent or trivalent ions replaceable forthe calcium ions can be removed from the BFP-like fluorescent protein bytreating with a chelating agent for sequestering calcium ions ordivalent or trivalent ions replaceable for the calcium ions.

The chelating agent used to produce the gFP-like protein in the presentinvention may be any agent but is not particularly limited, so long asit strongly binds to calcium ions or divalent or trivalent ionsreplaceable for the calcium ions. Examples of the chelating agentinclude ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis((3-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CyDTA),N-(2-hydroxyethyl)iminodiacetic acid (HIDA) and the like. As usedherein, the calcium ions or divalent or trivalent ions replaceable forthe calcium ions are the same as those described above.

The amount of the chelating agent used to produce the gFP-likefluorescent protein is not particularly limited unless its concentrationaffects regeneration of the gFP-like fluorescent protein. Since it isdemonstrated that 3 mols of calcium ions bind to 1 mol of ionapoaequorin, e.g., 3 mols or more are preferred.

In the method for producing the gFP-like protein, the reactiontemperature and reaction time are not particularly limited but aregenerally at 0° C. to 42° C. for 0.1 to 2 hours, at 4° C. to 37° C. for0.1 to 2 hours, or at 4° C. to 15° C. for 0.1 to 24 hours.

The gFP-like protein thus obtained may be further purified. Purificationof the gFP-like protein can be performed in a conventional manner ofseparation/purification. The separation/purification includes, forexample, precipitation with ammonium sulfate, gel filtrationchromatography, ion exchange chromatography, affinity chromatography,reversed phase high performance liquid chromatography, dialysis,ultrafiltration, etc., alone or in an appropriate combination of thesetechniques.

7.2.2. Use of Green Fluorescent Protein (gFP)-Like Protein

(1) Use as a Reporter Protein

The gFP-like protein of the present invention can also be used as areporter protein to determine the transcription activity of a promoter,etc. A polynucleotide encoding the apoprotein is fused to a targetpromoter or other expression control sequence (e.g., an enhancer, etc.)to construct a vector. The vector described above is transfected to ahost cell. Coelenteramide analogue of the present invention is broughtin contact with the host cell to produce the BFP-like protein, which isthen brought in contact with the chelating agent for removing calciumions or divalent or trivalent ions replaceable for the calcium ions,thereby to produce the gFP-like protein. By detecting the fluorescencefrom the gFP-like protein of the present invention, the activity of thetarget promoter or other expression control sequence can be determined.

(2) Use as a Detection Marker

The gFP-like protein of the present invention can be used as a detectionmarker by its fluorescence. The detection marker of the presentinvention can be used to detect a target substance in, e.g.,immunoassay, hybridization assay, etc. The gFP-like protein of thepresent invention can be used in the form bound to a target substance(protein, nucleic acid, etc.) in a conventional manner, includingchemical modification. Detection methods using the detection marker canbe performed in a conventional manner. The detection marker of thepresent invention can also be used to determine the distribution of atarget substance by expressing the marker, e.g., as a fusion protein ofthe apoprotein and the target substance, then inserting the fusionprotein into cells by means of microinjection or the like.Coelenteramide analogue of the present invention is brought in contacttherewith to produce the BFP-like protein, which is then brought incontact with the chelating agent for removing calcium ions or divalentor trivalent ions replaceable for the calcium ions to produce thegFP-like protein, and so on. The marker can thus be used to determinethe distribution of the target protein described above. The distributionof such a target protein, etc. can be determined by a method fordetection such as fluorescence imaging. The apoprotein can also be usedafter expression in cells, in addition to the insertion into cells bymeans of microinjection, etc

(3) Material for Amusement Supplies

The gFP-like protein of the present invention can be preferably used asa fluorescent material for amusement supplies. Examples of suchamusement supplies are fluorescent soap bubbles, fluorescent ice,fluorescent candies, fluorescent color paints, etc. The amusementsupplies of the present invention can be prepared in a conventionalmanner.

(4) Fluorescence Resonance Energy Transfer (FRET) Method

The gFP-like fluorescent protein of the present invention can be usedfor analyses including an analysis of biological functions, an assay forenzyme activities, etc., based on the principle of intermolecularinteraction by the fluorescence resonance energy transfer (FRET) method.

For example, using the gFP-like fluorescent protein of the invention asa donor or an acceptor and an organic compound or another fluorescentprotein as an acceptor or a donor, the interaction between the proteinscan be detected by causing fluorescence resonance energy transfer (FRET)between them. In some embodiments of the present invention, the organiccompound used as an acceptor or a donor is Hoechist 3342, Indo-1, DAP1,etc. In some other embodiments of the present invention, anotherfluorescent protein used as an acceptor or a donor is another greenfluorescent protein (GFP), another blue fluorescent protein (BFP),another mutant GFP fluorescent protein, phycobilin, etc. In a preferredembodiment of the present invention, the physiological functions to beanalyzed include an orphan receptor (in particular, G-protein conjugatedreceptor), apoptosis, transcription regulation by gene expression, etc.In a preferred embodiment of the present invention, the enzyme to beanalyzed is protease, esterase, kinase, etc.

Analysis of the physiological functions by the FRET method may beperformed by known methods, for example, by modifications of the methodsdescribed in Hoffmann, C. et al Nat. Methods (2005) 2: 171-176,Paulsson, J. F. et al. Exp. Diabetes Res. 2008:2008, 865850, etc. Assayfor the enzyme activity may be performed by known methods, for example,by modifications of the methods described in Ting, A. Y. et al (2001)Proc. Natl. Acad Sci. USA 98: 15003-15008, Evellin, S. et at (2004)Methods. Mol. Biol. 284: 259-270, Palmer A. E. & Tsien, R. Y. (2006)1:1057-1065, etc.

The present invention further provides a kit used for the analysismethod described above. The kit comprises the gFP-like fluorescentprotein of the present invention and the organic compound and/or anotherfluorescent protein. Reagent such as the gFP-like fluorescent protein ofthe present invention, organic compounds, other fluorescent proteins,etc. may be dissolved in a suitable solvent and prepared to be suitablefor storage. The solvent which may be used is at least one selected fromthe group consisting of water, ethanol, various buffer solutions, andthe like. The kit may additionally comprise, if necessary, at least oneselected from the group consisting of a container designed therefor,other necessary accessories and an instruction manual, and the like.

All literatures and publications mentioned in this specification areherein incorporated in their entirety by reference into thespecification, irrespective of their purposes. The disclosure of thespecification, the claims, abstract and drawings of Japanese ApplicationJP2010-088175 filed on Apr. 6, 2010, based upon which the presentapplication claims the benefit of priority, are entirely incorporatedherein by reference.

The objects, characteristics, advantages and of the present invention aswell as the idea thereof are apparent to those skilled in the art fromthe descriptions given herein, and those skilled in the art can easilyimplement the present invention. It is to be understood that the bestmode to carry out the invention and specific examples are to be taken aspreferred embodiments of the present invention. These descriptions areonly for illustrative and explanatory purposes and are not intended torestrict the invention thereto. It is further apparent to those skilledin the art that various modifications may be made based on thedescriptions given herein within the intent and scope of the presentinvention disclosed herein.

EXAMPLES Synthesis Examples Materials and Methods (1) High PerformanceLiquid Chromatography (HPLC)

The purities of coelenterazine analogues and coelenteramide analogueswere determined by a 1100 Series HPLC System manufactured by Agilent.The column used was a Lichrosorb RP-18 (5 μm, 4.0 mm i.d.×125 mm)manufactured by Merck Chemicals. Moving phase: gradient 60-100%methanol/0.1% aqueous TFA for 40 min; flow rate: 0.45 mL/min; detection:UV 225 nm; the amount of injected sample: 0.5 mg/5 mL in methanol/0.1%aqueous TFA=6/4.

(2) Chromatography

Thin layer chromatography (TLC) for analysis was performed on a glassplate (MERCK 5715, silica gel 60 F₂₅₄) previously coated with silicagel. The spots were detected under a UV lamp (254 nm or 365 nm) byadsorbing iodine, dipping in an aqueous anisaldehyde solution andcharring on a hot plate. The unsaturated C—C bonds were detected bydipping in an aqueous potassium permanganate solution followed bycharring on a hot plate.

For preparative flush column chromatography, silica gels (Kanto ChemicalCo., Inc., 37563-85, silica gel 60 N (spherical, neutral), particle size45-50 μm) and (Kanto Chemical Co., Inc., 37565-85, silica gel 60 N(spherical, neutral), particle size 63-210 μm) were used. Forpurification of the CTZ analogues, however, silica gels (Kanto ChemicalCo., Inc., 37562-79, silica gel 60 N (spherical), particle size 40-50μm) and (Kanto Chemical Co., Inc., 37558-79, silica gel 60 N(spherical), particle size 100-210 μm) were used.

(3) Nuclear Magnetic Resonance (NMR) Spectra

¹H Nuclear magnetic resonance (NMR) spectra (400 MHz) were determined ona Unity Plus 400 nuclear magnetic resonance apparatus manufactured byVarian, Inc. Chemical shifts (5) were expressed as values relative tothe peaks from tetramethylsilane ((CH₃)₄Si) (measured in CDCl₃; 0 ppm)or the peaks from non-deuterated solvent for analysis (measured inCD₃OD; 3.31 ppm, measured in DMSO-d₆; 2.49 ppm) as an internal standard.Abbreviations s, d and m used for the signal splitting patternsrepresent singlet, doublet and multiplet, respectively.

¹³C Nuclear magnetic resonance spectra (75.5 or 67.8 MHz) weredetermined by a Mercury 300 nuclear magnetic resonance apparatusmanufactured by Varian, Inc., or on JNM-EX270 manufactured by JEOL.Chemical shifts (5) were expressed as values relative to the peaks fromcarbon in the solvent for analysis (measured in acetone-d₆; 29.8 ppm,measured in DMSO-d₆; 39.5 ppm) as an internal standard.

(4) Infrared Absorption (IR) Spectra

IR spectra were determined by the diffuse reflection method using aSHIMADZU IR Prestige-21 spectrophotometer equipped with DRS-8000,manufactured by Shimadzu Corporation.

(5) Mass Spectrometry

High resolution mass spectrometry (HRMS) was performed by theelectrospray ionization method (ESI⁺) with a Bruker micrOTOFmanufactured by Bruker or by the fast atom bombardment method (FAB⁺)using JMS-700 manufactured by JEOL.

(6) Elemental Analysis

Elemental analysis (Anal.) was performed using YANACO CHN CORDER MT-5.

(7) Chemical Reagents

The reagents were all commercially available and used without furthertreatment unless otherwise indicated. The solvents for the reactions,extractions and chromatography were DMF, ethyl acetate, n-hexane,anhydrous THF, toluene, ethanol, DMSO, diethyl ether, methanol, THF,dichloromethane, 1,4-dioxane, anhydrous pyridine, diethylamine,anhydrous dichloromethane, anhydrous 1,2-dichloroethane and anhydrousDMF, all commercially available, and used without further treatment.

Mixing ratios of the solvents are based on volume, unless otherwiseindicated.

The reaction reagents below were used. Imidazole (Cat. No. 095-0015),triisopropyl borate (Cat. No. 324-41535), Na₂CO₃ (Cat. No. 199-01585),aminopyrazine (Cat. No. 013-12083), N-bromosuccinimide (Cat. No.025-07235), p-hydroxybenzaldehyde (Cat. No. 081-05925), triethylamine(Cat. No. 202-02646), methanesulfonyl chloride (Cat. No. 131-01583),1-naphthaleneboronic acid (Cat. No. 322-63393),benzo[b]thiophene-2-boronic acid (Cat. No. 329-64121),p-nitrophenylboronic acid (Cat. No. 327-59813), hydrochloric acid (Cat.No. 080-01066), p-hydroxyphenylacetic acid (Cat. No. 084-04185), oxalyldichloride (Cat. No. 155-01642), 4-(dimethylamino)pyridine (Cat. No.042-19212), thionyl chloride (Cat. No. 200-01106) and m-anisoyl chloride(Cat. No. 326-77701), purchased from Wako Pure Chemical Co., Ltd.,dichlorobis(triphenylphosphine) palladium (II) (Cat. No. 412740),2-naphthaleneboronic acid (Cat. No. 480134),2-(tributylstannyl)thiophene (Cat. No. 414492), thianaphthene-3-boronicacid (Cat. No. 512117), trans-2-phenylvinylboronic acid (Cat. No.473790), 3-thienylboronic acid (Cat. No. 436844), 1-phenylvinylboronicacid (Cat. No. 571350), 4-(dimethylamino)phenylboronic acid (Cat. No.483532), copper (I) iodide (Cat. No. 21554), phenylacetylene (Cat. No.117706) and 1 M tetrabutylammonium fluoride in THF (Cat. No. 216143),purchased from Aldrich, Inc., 4-bromophenol (Cat. No. B0787),tert-butyldimethylsilyl chloride (Cat. No. B0995),5-bromopyrazin-2-amine (Cat. No. A1683), ethyl diethoxyacetate (Cat. No.D1883), phenylboronic acid (Cat. No. B0857),thieno[3,2-b]thiophen-2-boronic acid (Cat. No. T2621),dithieno[3,2-b:2′,3′-d]thiophen-2-boronic acid (Cat. No. D3823),5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,2′-bithiophene (Cat.No. T2518), 4-methoxyphenyl isocyanate (Cat. No. 10440), 4-methoxyphenylisothiocyanate (Cat. No. 10513) and 4-methoxybenzoyl chloride (Cat. No.M0721), purchased from Tokyo Chemical Industry Co., Ltd., 2.64 M n-butyllithium in n-hexane (Cat. No. 04937-25), sodium borohydride (Cat. No.37828-35), magnesium, turning (Cat. No. 26000-25), NH₄Cl (Cat. No.01287-01) and sodium hydride (Cat. No. 37842-35), purchased from KantoChemical Co., Inc., and, palladium/charcoal activated (10% Pd) (Cat.8.07104.0010) purchased from MERCK, Inc., were used without furthertreatment.

Reference Synthesis Example 1 (4-Bromophenoxy)(tert-butyl)dimethylsilane(36) (known compound, Reference: P. Jeanjot, et al., Synthesis, 513-522(2003))

To a solution of 4-bromophenol (35) (20.0 g, 116 mmol) in DMF (200 mL)were successively added imidazole (23.6 g, 347 mmol) andtert-butyldimethylsilyl chloride (24.4 g, 162 mmol) at 0° C., and themixture was stirred for 3 hours while elevating to room temperature. Tothe mixture was added water and the product was extracted with ethylacetate (200 mL×3). The combined organic extract was washed successivelywith water (300 mL) and brine (200 mL), and dried over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 200 g,n-hexane/ethyl acetate=100/1) to give Compound 36 (32.3 g, crude) as acolorless oil. The product was used in the following reaction withoutfurther purification. R_(f)=0.57 (n-hexane/ethyl acetate=49/1); ¹H NMR(400 MHz, CDCl₃) δ 0.18 (s, 6H), 0.97 (s, 9H), 6.68-6.75 (AA′BB′, 2H),7.30-7.35 (AA′BB′, 2H).

Reference Synthesis Example 24-(tert-Butyldimethylsilyloxy)phenylboronic acid (37) (known compound,Reference: P. Jeanjot, et al., Synthesis, 513-522 (2003))

Under an argon atmosphere, to a solution of(4-bromophenoxy)(tert-butyl)dimethylsilane (36) (32.3 g, crude) preparedabove in anhydrous THF (300 mL) was added n-butyl lithium (2.64 Mn-hexane solution) (46.0 mL, 121 mmol) at −78° C., and the mixture wasstirred for an hour. To this was added triisopropyl borate (134 mL, 579mmol) at the same temperature and the mixture was stirred for 13 hourswhile elevating to room temperature. To the mixture was added 1 Mhydrochloric acid (200 mL) and the product was extracted with ethylacetate (200 mL×3). The combined organic extract was washed successivelywith water (300 mL) and brine (200 mL), followed by drying overanhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was recrystallized (n-hexane/ethylacetate) to give Compound 37 (20.9 g, 82.9 mmol, 71.8% (2 steps)) as acolorless solid. R_(f)=0.62 (n-hexane/ethyl acetate=1/1); ¹H NMR (400MHz, CDCl₃) δ 0.25 (s, 6H), 1.01 (s, 9H), 6.93-6.98 (AA′BB′, 2H),8.09-8.14 (AA′BB′, 2H).

Reference Synthesis Example 35-[4-(tert-Butyldimethylsilyloxy)phenyl]pyrazin-2-amine (39) (knowncompound)

Under an argon atmosphere, to a solution of4-(tert-butyldimethylsilyloxy)phenylboronic acid (37) (7.97 g, 31.6mmol) in toluene (290 mL) and ethanol (10 mL) were successively added5-bromopyrazin-2-amine (38) (5.00 g, 28.7 mmol),dichlorobis(triphenylphosphine)palladium (II) (1.21 g, 1.72 mmol) and 1M Na₂CO₃ aqueous solution (29.0 mL, 29.0 mmol) at room temperature, andthe mixture was heated to reflux for 17 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (100mL×3). The combined organic extract was washed successively with water(300 mL) and brine (200 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 200 g,n-hexane/ethyl acetate=2/1) to give Compound 39 (7.82 g, 25.9 mmol,90.2%) as a colorless solid. R_(f)=0.39 (n-hexane/ethyl acetate=1/1); ¹HNMR (400 MHz, CDCl₃) δ 0.22 (s, 6H), 1.00 (s, 9H), 4.53 (s, 2H),6.88-6.95 (AA′BB′, 2H), 7.71-7.79 (AA′BB′, 2H), 8.04 (s, 1H), 8.84 (s,1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C), 120.0(2C), 126.1 (2C), 130.7, 131.3, 138.1, 139.2, 154.5, 154.7; IR (KBr,cm⁻¹) 417, 478, 513, 544, 567, 629, 644, 658, 683, 750, 781, 806, 841,912, 1007, 1052, 1080, 1103, 1169, 1207, 1254, 1341, 1379, 1418, 1474,1506, 1539, 1570, 1605, 1639, 2857, 2893, 2928, 2955, 3163, 3302, 3431.

Reference Synthesis Example 43-Bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(known compound, Reference: F. D. Wael, et al., Bioorg. Med Chem., 17,4336-4344 (2009))

To a solution of 5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(39) (12.7 g, 42.1 mol) in DMSO (790 mL) and water (19.5 mL) was addedN-bromosuccinimide (7.90 g, 44.4 mmol) at room temperature and themixture was stirred for 17 hours. To the mixture was added water and theproduct was extracted with diethyl ether (400 mL×8). The combinedorganic extract was washed successively with water (300 mL×2) and brine(200 mL×2), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 200 g, n-hexane/ethylacetate=3/1) to give Compound 5 (13.0 g, 34.2 mmol, 80.9%) as a yellowsolid. R_(f)=0.32 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz, CDCl₃)δ 0.22 (s, 6H), 0.99 (s, 9H), 4.99 (s, 2H), 6.87-6.94 (AA′BB′, 2H),7.71-7.77 (AA′BB′, 2H), 8.34 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6(2C), 17.9, 25.4 (3C), 120.1 (2C), 124.7, 126.4 (2C), 128.9, 137.4,140.0, 152.0, 155.2; IR (KBr, cm⁻¹) 554, 619, 646, 675, 704, 756, 779,806, 849, 905, 1036, 1094, 1109, 1169, 1202, 1261, 1337, 1416, 1464,1501, 1566, 1605, 1630, 2857, 2928, 2955, 3138, 3281, 3460.

Reference Synthesis Example 5 3,5-Dibromopyrazin-2-amine (42) (knowncompound, Reference: P. Jeanjot, et al., Synthesis, 513-522 (2003))

To a solution of aminopyrazine (4l) (10.0 g, 105 mmol) in DMSO (200 mL)and water (5 mL) was added N-bromosuccinimide (39.3 g, 221 mmol) at roomtemperature, and the mixture was stirred for 23 hours. To the mixturewas added water and the product was extracted with diethyl ether (300mL×4). The combined organic extract was washed successively with water(400 mL×2) and brine (500 mL×2), followed by drying over anhydroussodium sulfate. After filtration and concentration under reducedpressure, the residue was purified by column chromatography (silica gel280 g, n-hexane/dichloromethane/ethyl acetate=5/4/1). The resultingsolid was further purified by recrystallization (n-hexane/ethyl acetate)to give Compound 42 (17.3 g, 68.3 mmol, 64.9%) as a colorless solid.R_(f)=0.48 (n-hexane/dichloromethane/ethyl acetate=5/4/1); ¹H NMR (400MHz, CDCl₃) δ 5.05 (s, 2H), 8.05 (s, 1H).

Reference Synthesis Example 6 4-(tert-Butyldimethylsilyloxy)benzaldehyde(44) (known compound, Reference: M. Adamczyk, et al., Tetrahedron, 59,8129-8142 (2003))

To a solution of p-hydroxybenzaldehyde (43) (12.0 g, 98.3 mmol) in DMF(25 mL) was successively added imidazole (16.7 g, 245 mmol) andtert-butyldimethylsilyl chloride (17.8 g, 118 mmol) at 0° C., and themixture was stirred for 3 hours while elevating to room temperature. Tothe mixture was added water and the product was extracted with ethylacetate (200 mL×3). The organic layer was washed successively withsaturated aqueous solution of K₂CO₃ (300 mL), water (300 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 200 g, n-hexane/ethylacetate=19/1) to give Compound 44 (20.5 g, 86.7 mmol, 88.2%) as acolorless oil. R_(f)=0.32 (n-hexane/ethyl acetate=19/1); ¹H NMR (400MHz, CDCl₃) δ 0.25 (s, 6H), 1.00 (s, 9H), 6.92-6.98 (AA′BB′, 2H),7.76-7.83 (AA′BB′, 2H), 9.89 (s, 1H).

Reference Synthesis Example 7 4-(tert-Butyldimethylsilyloxy)benzylalcohol (45) (known compound, Reference: M. Adamczyk, et al.,Tetrahedron, 59, 8129-8142 (2003))

To a solution of 4-(tert-butyldimethylsilyloxy)benzaldehyde (44) (20.5g,86.7 mmol) in methanol (120 mL) was added sodium borohydride (3.93 g,104 mmol) at 0° C., and the mixture was stirred for 5 hours whileelevating to room temperature. To the mixture was added water and theproduct was extracted with diethyl ether (300 mL×3). The combinedorganic extract was washed successively with water (300 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. The residuewas filtered and concentrated under reduced pressure to give Compound 45(20.5 g, 104 mmol, 100%) as a colorless oil. The product has asufficient purity and used in the following reaction without furtherpurification. R_(f)=0.21 (n-hexane/ethyl acetate=19/1); ¹H NMR (400 MHz,CDCl₃) δ 0.19 (s, 6H), 0.98 (s, 9H), 1.59 (br, 1H), 4.61 (s, 2H),6.80-6.85 (AA′BB′, 2H), 7.20-7.26 (AA′BB′, 2H).

Reference Synthesis Example 8 4-(tert-Butyldimethylsilyloxy)benzylchloride (46) (known compound, Reference: M. Adamczyk, et al.,Tetrahedron, 59, 8129-8142 (2003))

To a solution of 4-(tert-butyldimethylsilyloxy)benzyl alcohol (45) (10.0g, 42.0 mmol) in DMF (100 mL) were successively added triethylamine(12.0 mL, 86.1 mmol) and methanesulfonyl chloride (4.90 mL, 63.1 mmol)at 0° C., and the mixture was stirred for 20 hours while elevating toroom temperature. To the mixture was added water and the product wasextracted with ethyl acetate (200 mL×3). The combined organic extractwas washed successively with water (300 mL) and brine (200 mL), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by columnchromatography (silica gel 200 g, n-hexane/diethyl ether=10/1) to giveCompound 46 (8.07 g, 31.4 mmol, 74.9%) as a colorless oil. R_(f)=0.71(n-hexane/ethyl acetate=19/1); ¹H NMR (400 MHz, CDCl₃) δ 0.20 (s, 6H),0.98 (s, 9H), 4.56 (s, 2H), 6.78-6.84 (AA′BB′, 2H), 7.22-7.27 (AA′BB′,2H).

Reference Synthesis Example 93-[4-(tert-Butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(known compound, Reference: M. Adamczyk, et al., Tetrahedron, 59,8129-8142 (2003))

Magnesium turnings (640 mg, 26.3 mmol) were placed in a reaction flaskand dried in vacuo by heating with a heat gun. The flask were thenallowed to cool to room temperature and filled with a dry argonatmosphere. To this was added anhydrous THF (28 mL) and to the mixturewas added dropwise 4-(tert-butyldimethylsilyloxy)benzyl chloride (46)(6.15 g, 24.0 mmol) at room temperature. The mixture was stirred at roomtemperature for an hour while the most of the magnesium turningsdisappeared affording a THF solution of4-(tert-butyldimethylsilyloxy)benzyl magnesium chloride, which was usedin the following reaction without further treatment.

Under an argon atmosphere, to a solution of ethyl diethoxyacetate (4.30mL, 24.0 mmol) in anhydrous THF (50 mL) was added the THF solution of4-(tert-butyldimethylsilyloxy)benzyl magnesium chloride prepared aboveat −78° C. The mixture was stirred at −78° C. for 2 hours and an aqueous10% NH₄Cl solution (150 mL) was added thereto. The product was extractedwith ethyl acetate (200 mL×3). The combined organic extract was washedsuccessively with a 10% NH₄Cl aqueous solution (300 mL), water (300 mL)and brine (200 mL), followed by drying over anhydrous sodium sulfate.After filtration and concentration under reduced pressure, the residuewas purified by column chromatography (silica gel 200 g, n-hexane/ethylacetate=40/1) to give Compound 8 (6.19 g, 17.6 mmol, 72.9%) as acolorless oil. R_(f)=0.43 (n-hexane/ethyl acetate=19/1); ¹H NMR (400MHz, CDCl₃) δ 0.18 (s, 6H), 0.97 (s, 9H), 1.24 (t, 6H, J=7.1 Hz), 3.53(dq, 2H, J=2.3, 7.1 Hz), 3.68 (dq, 2H, J=2.3, 7.1 Hz), 4.63 (s, 1H),6.74-6.81 (AA′BB′, 2H), 7.03-7.10 (AA′BB′, 2H).

Reference Synthesis Example 102-[4-(tert-Butyldimethylsilyloxy)phenyl]acetic acid (9) (known compound,Reference: O. Brummer, et al., Tetrahedron Lett., 42, 2257-2259 (2001))

To a solution of p-hydroxyphenylacetic acid (48) (10.0 g, 65.7 mmol) inTHF (80 mL) were successively added imidazole (22.4 g, 329 mmol) andtert-butyldimethylsilyl chloride (27.7 g, 184 mmol) at 0° C., and themixture was stirred for an hour while elevating to room temperature. Tothis was added a solution of saturated Na₂CO₃ (250 mL) and the mixturewas stirred at room temperature for an hour. To the mixture was added 2M HCl aqueous solution (650 mL) and the product was extracted with ethylacetate (300 mL×3). The combined organic extract was washed successivelywith water (400 mL) and brine (300 mL), followed by drying overanhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 200 g, n-hexane→n-hexane/ethyl acetate/acetic acid=1/1/0.05)to give Compound 9 (10.6 g, 39.9 mmol, 60.7%) as a colorless solid.R_(f)=0.64 (n-hexane/ethyl acetate=1/2); ¹H NMR (400 MHz, CDCl₃) δ 0.20(s, 6H), 0.99 (s, 9H), 3.58 (s, 2H), 6.76-6.85 (AA′BB′, 2H), 7.10-7.17(AA′BB′, 2H).

Synthesis Example 1 Coelenterazine (CTZ) analogues modified at the C-8position 1-1) TMD-296 (3a)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-phenylpyrazin-2-amine (7a)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.50 g, 3.94 mmol) in toluene (45 mL) and ethanol (1.7 mL) weresuccessively added phenylboronic acid (6a) (577 mg, 4.73 mmol),dichlorobis(triphenylphosphine)palladium (II) (165 mg, 235 μmol) and 1 MNa₂CO₃ aqueous solution (4.00 mL, 4.00 mmol) at room temperature, andthe mixture was heated to reflux for 17 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (200mL×3). The combined organic extract was washed successively with water(300 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 100 g,n-hexane/ethyl acetate=3/1) to give Compound 7a (1.46 g, 3.87 mmol,98.1%) as a brown solid. R_(f)=0.41 (n-hexane/ethyl acetate=1/1); ¹H NMR(400 MHz, DMSO-d₆) 0.21 (s, 6H), 0.96 (s, 9H), 6.20 (s, 2H), 6.89-6.94(AA′BB′, 2H), 7.42-7.55 (m, 3H), 7.76-7.82 (m, 2H), 7.85-7.91 (AA′BB′,2H), 8.48 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (2C), 17.9, 25.5(3C), 120.0 (2C), 126.4 (2C), 128.2 (2C), 128.4, 128.6 (2C), 130.5,137.2, 137.68, 137.72, 139.9, 151.6, 155.0; IR (KBr, cm⁻¹) 523, 637,677, 700, 720, 752, 779, 808, 841, 914, 1018, 1070, 1092, 1105, 1167,1204, 1263, 1319, 1362, 1381, 1435, 1460, 1510, 1605, 2857, 2886, 2928,2955, 3059, 3159, 3291, 3428; HRMS (ESI m/z 378.1990 ([M+H]⁺,C₂₂H₂₈N₃OSi⁺ requires 378.1996).

2-(4-Hydroxybenzyl)-6-(4-hydroxyphenyl)-8-phenylimidazo[1,2-a]pyrazin-3(7H)-one(3a, TMD-296)

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-phenylpyrazin-2-amine (7a)(272 mg, 720 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(381 mg, δ 08 mmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (780 μL) at 0° C., and the mixture was heated toreflux for 17 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3a (TMD-296) (158 mg, 386μmol, 53.6%) as an orange solid. R_(f)=0.59 (ethyl acetate); HPLCretention time 8.0 min; ¹H NMR (400 MHz, CD₃OD) δ 4.16 (s, 2H),6.69-6.76 (AA′BB′, 2H), 6.91-6.97 (AA′BB′, 2H), 7.09-7.16 (AA′BB′, 2H),7.56-7.68 (m, 3H), 7.84-7.91 (AA′BB′, 2H), 7.98-8.03 (m, 2H), 8.39 (s,1H); IR (KBr, cm⁻¹) 523, 656, 696, 779, 843, 891, 1173, 1265, 1342,1443, 1512, 1591, 1609, 1655, 2814, 3154; HRMS (ESI⁺) m/z 410.1497([M+H]⁺, C₂₅H₂₀N₃O₃ ⁺ requires 410.1499). 1-2) TMD-282 (3b)

(E)-5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-styrylpyrazin-2-amine(7b)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.00 g, 2.63 mmol) in toluene (30 mL) and ethanol (1.2 mL) weresuccessively added trans-2-phenylvinylboronic acid (6b) (543 mg, 3.16mmol), dichlorobis(triphenylphosphine)palladium (II) (111 mg, 158 μmol)and 1 M Na₂CO₃ aqueous solution (2.70 mL, 2.70 mmol) at roomtemperature, and the mixture was heated to reflux for 18 hours. Aftercooling to room temperature, to the mixture was added water, and themetal catalyst was removed by filtration. The product was extracted withethyl acetate (200 mL×3). The combined organic extract was washedsuccessively with water (300 mL) and brine (300 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 100 g, n-hexane/ethyl acetate=3/1) to give Compound 7b (997mg, 2.47 mmol, 94.0%) as a yellow solid. R_(f)=0.19 (n-hexane/ethylacetate=3/1); ¹H NMR (400 MHz, DMSO-d₆) δ 0.19 (s, 6H), 0.94 (s, 9H),6.60 (s, 2H), 6.84-6.91 (AA′BB′, 2H), 7.22-7.31 (m, 1H), 7.31-7.44 (m,2H), 7.55 (d, 1H, J=16 Hz), 7.65-7.77 (m, 3H), 7.87-7.93 (AA′BB′, 2H),9.39 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C),120.1 (2C), 122.1, 126.5 (2C), 127.3 (2C), 128.2, 128.7 (2C), 130.8,132.8, 133.8, 136.9, 137.7, 139.4, 152.0, 155.0; IR (KBr, cm⁻¹) 444,463, 507, 534, 633, 691, 745, 781, 806, 841, 914, 964, 1011, 1072, 1088,1103, 1153, 1213, 1263, 1379, 1420, 1454, 1512, 1566, 1605, 1634, 2856,2886, 2930, 2955, 3057, 3196, 3329; HRMS (ESI⁺) m/z 404.2161 ([M+H]⁺,C₂₄H₃₀N₃OSi⁺ requires 404.2153).

(E)-2-(4-Hydroxybenzyl)-6-(4-hydroxyphenyl)-8-styrylimidazo[1,2-a]pyrazin-3(7H)-one(3b, TMD-282)

Under an argon atmosphere, to a mixture of(E)-5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-styrylpyrazin-2-amine(7b) (290 mg, 719 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(380 mg, 1.08 mmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (600 μL) at, 0° C. and the mixture was heated toreflux for 15 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3b (TMD-282) (74.0 mg,170 μmol, 23.6%) as a brown solid. R_(f)=0.22 (ethyl acetate); HPLCretention time 14.3 min. 1-3) TMD-276 (3d)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(naphthalen-1-yl)pyrazin-2-amine(7d)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.48 g, 3.90 mmol) in toluene (45 mL) and ethanol (2 mL) weresuccessively added 1-naphthaleneboronic acid (6d) (805 mg, 4.68 mmol),dichlorobis(triphenylphosphine)palladium (II) (164 mg, 234 μmol) and 1 MNa₂CO₃ aqueous solution (4.00 mL, 4.00 mmol) at room temperature, andthe mixture was heated to reflux for 18 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (200mL×3). The combined organic extract was washed successively with water(300 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 100 g,n-hexane/ethyl acetate=4/1) to give Compound 7d (1.44 g, 3.37 mmol,86.5%) as a yellow foamy solid. R_(f)=0.25 (n-hexane/ethyl acetate=4/1);¹H NMR (400 MHz, DMSO-d₆) δ 0.14 (s, 6H), 0.90 (s, 9H), 5.89 (s, 2H),6.80-6.87 (AA′BB′, 2H), 7.41-7.48 (m, 1H), 7.48-7.64 (m, 4H), 7.74-7.81(AA′BB′, 2H), 7.96-8.04 (m, 2H), 8.55 (s, 1H); ¹³C NMR (75.5 MHz,DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C), 120.1 (2C), 125.2, 125.8, 126.0,126.37 (2C), 126.40, 127.2, 128.4, 128.8, 130.6, 130.9, 133.6, 134.7,137.8, 138.1, 139.5, 152.5, 154.9; IR (KBr, cm⁻¹) 517, 544, 579, 631,675, 706, 739, 777, 806, 841, 910, 980, 1055, 1080, 1103, 1121, 1169,1179, 1198, 1265, 1337, 1362, 1375, 1423, 1450, 1508, 1570, 1605, 2857,2886, 2928, 2955, 3051, 3179, 3300, 3383, 3474; HRMS (ESI⁺) m/z 428.2163([M+H]⁺, C₂₆H₃₀N₃OSi⁺ requires 428.2153).

2-(4-Hydroxybenzyl)-6-(4-hydroxyphenyl)-8-(naphthalen-1-yl)imidazo[1,2-a]pyrazin-3(7H)-one(3d, TMD-276)

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(naphthalen-1-yl)pyrazin-2-amine(7d) (255 mg, 596 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(315 mg, 894 μmol) dissolved in 1,4-dioxane (2 mL) was added, 4 Mhydrochloric acid (780 μL) at 0° C. and the mixture was heated to refluxfor 15 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3d (TMD-276) (74.3 mg,162 mol, 27.1%) as an orange solid. R_(f)=0.54 (ethylacetate/methanol=20/1); HPLC retention time 9.1 min; ¹H NMR (400 MHz,CD₃OD) δ 4.06 (s, 2H), 6.64-6.70 (AA′BB′, 2H), 6.88-695 (AA′BB′, 2H),7.00-7.07 (AA′BB′, 2H), 7.47-7.55 (m, 1H), 7.55-7.63 (m, 1H), 7.68-7.73(m, 1H), 7.80-7.92 (m, 4H, includes AA′BB′), 8.00-8.07 (m, 1H),8.14-8.22 (m, 1H), 8.54 (s, 1H); IR (KBr, cm⁻¹) 463, 492, 646, 779, 808,968, 1015, 1045, 1082, 1109, 1173, 1240, 1325, 1346, 1443, 1508, 1591,1609, 1653, 2814, 3169; HRMS (ESI⁺) m/z 460.1656 ([M+H]⁺, C₂₉H₂₂N₃O₃ ⁺requires 460.1656).

1-4) TMD-277 (3e)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(naphthalen-2-yl)pyrazin-2-amine(7e)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.00 g, 2.63 mmol) in toluene (30 mL) and ethanol (1.2 mL) weresuccessively added 2-naphthaleneboronic acid (6e) (543 mg, 3.16 mmol),dichlorobis(triphenylphosphine)palladium (II) (110 mg, 157 μmol) and 1 MNa₂CO₃ aqueous solution (2.70 mL, 2.70 mmol) at room temperature, andthe mixture was heated to reflux for 15 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (200mL×3). The combined organic extract was washed successively with water(300 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 100 g,n-hexane/ethyl acetate=3/1) to give Compound 7e (1.04 g, 2.43 mmol,92.2%) as a yellow solid. R_(f)=0.33 (n-hexane/diethyl ether=2/3); ¹HNMR (400 MHz, DMSO-d₆) δ 0.17 (s, 6H), 0.92 (s, 9H), 6.32 (s, 2H),6.85-6.92 (AA′BB′, 2H), 7.49-7.57 (m, 2H), 7.84-7.91 (m, 3H, includesAA′BB′), 7.91-7.96 (m, 1H), 7.97-8.03 (m, 2H), 8.31 (s, 1H), 8.47 (s,1H); IR (KBr, cm⁻¹) 419, 438, 530, 648, 669, 698, 743, 779, 814, 841,918, 939, 1009, 1103, 1126, 1165, 1192, 1219, 1252, 1341, 1362, 1389,1420, 1431, 1462, 1508, 1530, 1566, 1605, 1636, 2859, 2895, 2930, 2955,3038, 3057, 3159, 3296, 3416; HRMS (ESI⁺) m/z 428.2155 ([M+H]⁺,C₂₆H₃₀N₃OSi⁺ requires 428.2153).

2-(4-Hydroxybenzyl)-6-(4-hydroxyphenyl)-8-(naphthalen-2-yl)imidazo[1,2-a]pyrazin-3(7H)-one (3e, TMD-277)

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(naphthalen-2-yl)pyrazin-2-amine(7e) (308 mg, 720 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(381 mg, 1.08 mmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (600 μL) at 0° C., and the mixture was heated toreflux for 14 hours. After cooling to room temperature the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3e (TMD-277) (89.9 mg,196 μmol, 27.2%) as an orange solid. R_(f)=0.59 (ethylacetate/methanol=20/1); HPLC retention time 13.6 min; ¹H NMR (400 MHz,CD₃OD) δ 4.17 (s, 2H), 6.70-6.77 (AA′BB′, 2H), 6.91-6.98 (AA′BB′, 2H),7.11-7.17 (AA′BB′, 2H), 7.56-7.68 (m, 2H), 7.88-7.94 (AA′BB′, 2H),7.95-8.00 (m, 1H), 8.01-8.06 (m, 1H), 8.08-8.11 (m, 2H), 8.37 (s, 1H),8.55 (s, 1H); IR (KBr, cm⁻¹) 480, 552, 656, 752, 822, 903, 1111, 1173,1242, 1269, 1356, 1447, 1513, 1558, 1611, 1653, 2814, 2897, 3165; HRMS(ESI⁺) m/z 460.1666 ([M+H]⁺, C₂₉H₂₂N₃O₃ ⁺ requires 460.1656).

1-5) TMD-278 (3f)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(thiophen-2-yl)pyrazin-2-amine(7f)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.20 g, 3.15 mmol) in 1,4-dioxane (30 mL) were successively added2-(tributylstannyl)thiophene (6f) (1.10 mL, 3.46 mmol) anddichlorobis(triphenylphosphine)palladium (II) (111 mg, 158 μmol) at roomtemperature, and the mixture was heated to reflux for 14 hours. Aftercooling to room temperature, to the mixture was added saturated KFaqueous solution and the mixture was stirred at room temperature for 30minutes. The metal catalyst was removed by filtration, and the productwas extracted with ethyl acetate (200 mL×3). The combined organicextract was washed successively with saturated KF aqueous solution (300mL), water (300 mL) and brine (300 mL), followed by drying overanhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 100 g, n-hexane/ethyl acetate=3/1) to give Compound 7f (1.03g, 2.68 mmol, 84.9%) as a yellow solid. R_(f)=0.33 (n-hexane/ethylacetate=3/1); ¹H NMR (400 MHz, DMSO-d₆) δ 0.19 (s, 6H), 0.94 (s, 9H),6.42 (s, 2H), 6.87-6.94 (AA′BB′, 2H), 7.17 (dd, 1H, J=3.8, 5.2 Hz), 7.65(d, 1H, J=5.2 Hz), 7.74 (d, 1H, J=3.8 Hz), 7.83-7.91 (AA′BB′, 2H), 8.46(s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C), 120.1(2C), 125.6, 126.4 (2C), 128.2, 128.3, 129.9, 131.8, 136.9, 139.5,142.6, 149.8, 155.2; IR (KBr, cm⁻¹) 471, 509, 536, 581, 623, 646, 669,716, 741, 779, 804, 822, 839, 912, 1011, 1057, 1082, 1111, 1172, 1202,1263, 1279, 1360, 1379, 1435, 1454, 1477, 1512, 1564, 1607, 2857, 2895,2930, 2953, 3335, 3439; HRMS (ESI⁺) m/z 384.1551 ([M+H]⁺, C₂₀H₂₆N₃OSSi⁺requires 384.1560).

2-(4-Hydroxybenzyl)-6-(4-hydroxyphenyl)-8-(thiophen-2-yl)imidazo[1,2-a]pyrazin-3(7H)-one(3f, TMD-278)

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thiophen-2-yl)pyrazin-2-amine(7f) (276 mg, 720 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(381 mg, 1.08 mmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (780 μL) at 0° C. and the mixture was heated to refluxfor 16 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3f (TMD-278) (68.2 mg,164 μmol, 22.8%) as a brown solid. R_(f)=0.67 (ethylacetate/methanol=20/1); HPLC retention time 9.5 min; ¹H NMR (400 MHz,CD₃OD) δ 4.22 (s, 2H), 6.72-6.78 (AA′BB′, 2H), 6.90-6.97 (AA′BB′, 2H),7.11-7.18 (AA′BB′, 2H), 7.32 (dd, 1H, J=3.8, 5.2 Hz), 7.87 (dd, 1H,J=1.1, 5.2 Hz), 7.91-7.97 (AA′BB′, 2H), 8.12 (dd, 1H, J=1.1, 3.8 Hz),8.48 (s, 1H); IR (KBr, cm⁻¹) 521, 581, 629, 721, 777, 841, 881, 966,1109, 1172, 1261, 1341, 1429, 1508, 1533, 1609, 1653, 2822, 3107; HRMS(ESI⁺) m/z 416.1075 ([M+H]⁺, C₂₃H₁₈N₃O₃S⁺ requires 416.1063).

1-6) TMD-336 (3g)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(thiophen-3-yl)pyrazin-2-amine(7g)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.50 g, 3.94 mmol) in toluene (45 mL) and ethanol (1.8 mL) weresuccessively added 3-thienylboronic acid (6g) (605 mg, 4.73 mmol),dichlorobis(triphenylphosphine)palladium (II) (165 mg, 235 μmol) and 1 MNa₂CO₃ aqueous solution (4.00 mL, 4.00 mmol) at room temperature and themixture was heated to reflux for 15 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (200mL×3). The combined organic extract was washed successively with water(300 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 150 g,n-hexane/ethyl acetate=3/1) to give Compound 7g (1.19 g, 3.11 mmol,78.9%) as a red solid. R_(f)=0.50 (n-hexane/ethyl acetate=1/1); ¹H NMR(400 MHz, DMSO-d₆) δ 0.21 (s, 6H), 0.96 (s, 9H), 6.29 (s, 2H), 6.88-6.96(AA′BB′, 2H), 7.66-7.73 (m, 2H), 7.88-7.93 (AA′BB′, 2H), 8.06-8.09 (m,1H), 8.47 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-dc) δ −4.5 (2C), 17.9, 25.5(3C), 120.1 (2C), 124.7, 126.1, 126.4 (2C), 128.2, 130.4, 133.5, 136.7,138.7, 139.6, 151.2, 155.0; IR (KBr, cm⁻¹) 511, 534, 551, 635, 662, 694,719, 737, 754, 779, 808, 843, 916, 1009, 1034, 1086, 1103, 1167, 1182,1196, 1215, 1260, 1331, 1366, 1389, 1408, 1454, 1510, 1566, 1605, 2857,2886, 2928, 2953, 3038, 3115, 3175, 3289, 3412; HRMS (ESI⁺) m/z 384.1560([M+H]⁺, C₂₀H₂₆N₃OSSi⁺ requires 384.1560).

2-(4-Hydroxybenzyl)-6-(4-hydroxyphenyl)-8-(thiophen-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one(3g, TMD-336)

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thiophen-3-yl)pyrazin-2-amine(7g) (311 mg, 811 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(396 mg, 1.22 mmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (780 μL) at 0° C., and the mixture was heated toreflux for 15 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3g (TMD-336) (179 mg, 431μmol, 53.2%) as an orange solid. R_(f)=0.70 (ethyl acetate); HPLCretention time 7.9 min; ¹H NMR (400 MHz, CD₃OD) δ 4.18 (s, 2H),6.70-6.76 (AA′BB′, 2H), 6.91-6.99 (AA′BB′, 2H), 7.11-7.19 (AA′BB′, 2H),7.70 (dd, 1H, J=2.8, 5.2 Hz), 7.81-7.88 (AA′BB′, 2H), 7.95 (dd, 1H,J=1.2, 5.2 Hz), 8.29 (s, 1H), 8.50 (dd, 1H, J=1.2, 2.8 Hz); IR (KBr,cm⁻¹) 519, 652, 804, 841, 926, 1173, 1233, 1271, 1341, 1437, 1508, 1609,1647, 3107; HRMS (ESI⁺) m/z 416.1065 ([M+H]⁺, C₂₃H₁₈N₃O₃S⁺ requires416.1063).

1-7) TMD-281 (3h)

3-(Benzo[b]thiophen-2-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7h)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (971mg, 2.55 mmol) in toluene (27 mL) and ethanol (1.3 mL) were successivelyadded benzo[b]thiophene-2-boronic acid (6h) (500 mg, 2.81 mmol),dichlorobis(triphenylphosphine)palladium (II) (108 mg, 153 μmol) and 1 MNa₂CO₃ aqueous solution (2.60 mL, 2.60 mmol) at room temperature and themixture was heated to reflux for 17 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (200mL×3). The combined organic extract was washed successively with water(300 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 100 g,n-hexane/ethyl acetate=3/1) to give Compound 7h (746 mg, 1.72 mmol,67.3%) as an orange solid. R_(f)=0.67 (n-hexane/ethyl acetate=1/1); ¹HNMR (400 MHz, DMSO-d₆) δ 0.20 (s, 6H), 0.95 (s, 9H), 6.68 (s, 2H),6.91-6.98 (AA′BB′, 2H), 7.33-7.41 (m, 2H), 7.81-7.88 (m, 1H), 7.91-7.99(m, 3H, includes AA′BB′), 8.11 (s, 1H), 8.55 (s, 1H); ¹³C NMR (75.5 MHz,DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C), 120.2 (2C), 122.0, 122.3, 124.37,124.42, 125.1, 126.5 (2C), 129.8, 131.0, 137.8, 139.4, 139.6, 140.9,143.1, 150.4, 155.3; IR (KBr, cm⁻¹) 527, 552, 584, 642, 675, 708, 723,741, 779, 808, 835, 918, 974, 1009, 1072, 1101, 1128, 1165, 1221, 1252,1371, 1418, 1462, 1510, 1533, 1566, 1605, 1636, 1728, 2857, 2895, 2928,2955, 3161, 3294, 3416; HRMS (ESI⁺) m/z 434.1713 ([M+H]⁺, C₂₄H₂₈N₃OSSi⁺requires 434.1717).

8-(Benzo[b]thiophen-2-yl)-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one(3h, TMD-281)

Under an argon atmosphere, to a mixture of3-(benzo[b]thiophen-2-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7h) (153 mg, 352 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(190 mg, 539 μmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (400 μL) at 0° C. and the mixture was heated to refluxfor 14 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3h (TMD-281) (31.0 mg,66.6 μmol, 18.9%) as an orange solid. R_(f)=0.67 (ethylacetate/methanol=20/1); HPLC retention time 19.9 min; ¹H NMR (400 MHz,CD₃OD) δ 4.12 (s, 2H), 6.75-6.84 (AA′BB′, 2H), 6.84-6.94 (AA′BB′, 2H),7.16-7.24 (AA′BB′, 2H), 7.32-7.45 (m, 2H), 7.77-7.84 (m, 2H), 7.85-7.92(AA′BB′, 2H), 8.30 (s, 1H), 8.35 (s, 1H); IR (KBr, cm⁻¹) 430, 523, 573,621, 637, 658, 725, 746, 839, 885, 962, 1107, 1173, 1204, 1233, 1342,1441, 1519, 1560, 1611, 1655, 3057.

1-8) TMD-337 (3i)

3-(Benzo[b]thiophen-3-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7i)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.50 g, 3.94 mmol) in toluene (45 mL) and ethanol (1.8 mL) weresuccessively added thianaphthene-3-boronic acid (6i) (842 mg, 4.73mmol), dichlorobis(triphenylphosphine)palladium (II) (165 mg, 235 μmol)and 1 M Na₂CO₃ aqueous solution (4.00 mL, 4.00 mmol) at roomtemperature, and the mixture was heated to reflux for 15 hours. Aftercooling to room temperature, to the mixture was added water and themetal catalyst was removed by filtration. The product was extracted withethyl acetate (200 mL×3). The combined organic extract was washedsuccessively with water (300 mL) and brine (300 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 150 g, n-hexane/ethyl acetate=3/1) to give Compound 7i (1.19g, 3.11 mmol, 78.9%) as an orange solid. R_(f)=0.30 (n-hexane/diethylether=2/3); ¹H NMR (400 MHz, DMSO-d₆) δ 0.21 (s, 6H), 0.96 (s, 9H), 6.29(s, 2H), 6.89-6.95 (AA′BB′, 2H), 7.41-7.92 (m, 2H), 7.85-7.92 (AA′BB′,2H), 7.95-8.02 (m, 1H), 8.07-8.13 (m, 1H), 8.16 (s, 1H), 8.55 (s, 1H);¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C), 120.1 (2C),122.8, 123.6, 124.4, 124.6, 126.3 (2C), 127.7, 130.4, 132.2, 133.7,137.3, 138.0, 139.3, 139.7, 152.5, 155.0, IR (KBr, cm⁻¹) 471, 631, 664,683, 712, 733, 758, 781, 808, 839, 912, 968, 1011, 1059, 1080, 1103,1128, 1169, 1188, 1263, 1346, 1362, 1389, 1447, 1508, 1605, 2857, 2886,2928, 2953, 3065, 3177, 3298, 3368, 3472; HRMS (ESI⁺) m/z 434.1725([M+H], C₂₄H₂₈N₃OSSi⁺ requires 434.1769).

8-(Benzo[b]thiophen-3-yl)-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one(3i, TMD-337)

Under an argon atmosphere, to a mixture of3-(benzo[b]thiophen-3-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7i) (336 mg, 775 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(378 mg, 1.16 mmol) in 1,4-dioxane (2 mL) was added 4 M hydrochloricacid (780 μL) at 0° C. and the mixture was heated to reflux for 18hours. After cooling to room temperature, the mixture was concentratedunder reduced pressure and the residue was purified by columnchromatography in an argon flow (acidic silica gel 20 g, n-hexane/ethylacetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1). To thesuspension obtained during the concentration under reduced pressure wasadded n-hexane. The precipitates were collected by filtration and driedin vacuo to give Compound 3i (TMD-337) (136 mg, 292 μmol, 37.6%) as anorange solid. R_(f)=0.37 (ethyl acetate); HPLC retention time 11.5 min;¹H NMR (400 MHz, CD₃OD) δ 4.16 (s, 2H), 6.68-6.77 (AA′BB′, 2H),6.90-6.96 (AA′BB′, 2H), 7.07-7.15 (AA′BB′, 2H), 7.43-7.54 (m, 2H),7.90-7.97 (AA′BB′, 2H), 8.00-8.07 (m, 1H), 8.22-8.30 (m, 1H), 8.36 (s,1H), 8.52 (s, 1H); IR (KBr, cm⁻¹) 517, 571, 637, 735, 764, 839, 959,1049, 1107, 1171, 1231, 1354, 1362, 1437, 1508, 1608, 3101; HRMS (ESI⁺)m/z 466.1233 ([M+H]⁺, C₂₇H₂₀N₃O₃S⁺ requires 466.1220).

1-9) TMD-280 (3j)

3,5-Bis[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (7j) (knowncompound, Reference: P. Jeanjot, et al., Synthesis, 513-522 (2003))

Under an argon atmosphere, to a solution of 3,5-dibromopyrazin-2-amine(42) (1.00 g, 3.95 mmol) dissolved in toluene (60 mL) and ethanol (4.0mL) were successively added (4-bromophenoxy)(tert-butyl)dimethylsilane(6j) (2.49 g, 9.89 mmol), dichlorobis(triphenylphosphine)palladium (II)(167 mg, 237 μmol) and 1 M Na₂CO₃ aqueous solution (8.00 mL, 8.00 mmol)at room temperature, and the mixture was heated to reflux for 17 hours.After cooling to room temperature, to the mixture was added water andthe metal catalyst was removed by filtration. The product was extractedwith ethyl acetate (200 mL×3). The combined organic extract was washedsuccessively with water (300 mL) and brine (300 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 100 g, n-hexane/ethyl acetate=4/1) to give Compound 7j (1.75g, δ 45 mmol, 87.4%) as a yellow solid. R_(f)=0.26 (n-hexane/ethylacetate=4/1); ¹H NMR (400 MHz, DMSO-dc) δ 0.18 (s, 6H), 0.22 (s, 6H),0.93 (s, 9H), 0.96 (s, 9H), 6.11 (s, 2H), 6.85-6.91 (AA′BB′, 2H),6.91-6.97 (AA′BB′, 2H), 7.65-7.71 (AA′BB′, 2H), 7.81-7.87 (AA′BB′, 2H),8.40 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.5 (4C), 18.0 (2C), 25.6(6C), 119.9 (2C), 120.1 (2C), 126.4 (2C), 129.6 (2C), 130.6, 130.8,136.6, 137.7, 139.8, 151.5, 154.9, 155.5, IR (KBr, cm⁻¹) 438, 513, 573,633, 665, 675, 696, 739, 781, 806, 824, 841, 912, 1009, 1086, 1103,1167, 1202, 1263, 1362, 1379, 1408, 1422, 1452, 1512, 1566, 1605, 2857,2885, 2928, 2955, 3061, 3183, 3304, 3377, 3478; HRMS (ESI⁺) m/z 508.2820([M+H]⁺, C₂₈H₄₂N₃O₂Si₂ ⁺ requires 508.2810).

2-(4-Hydroxybenzyl)-6,8-bis(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one(3j, TMD-280)

Under an argon atmosphere, to a mixture of3,5-bis[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (7j) (366mg, 721 μmol) and3-[4-(tert-butyldimethylsilyloxy)phenyl]-1,1-diethoxypropan-2-one (8)(380 mg, 1.08 mmol) dissolved in 1,4-dioxane (2 mL) was added 4 Mhydrochloric acid (900 μL) at 0° C., and the mixture was heated toreflux for 16 hours. After cooling to room temperature, the mixture wasconcentrated under reduced pressure, and the residue was purified bycolumn chromatography in an argon flow (acidic silica gel 20 g,n-hexane/ethyl acetate=1/2→ethyl acetate→ethyl acetate/methanol=20/1).To the suspension obtained during the concentration under reducedpressure was added n-hexane. The precipitates were collected byfiltration and dried in vacuo to give Compound 3j (TMD-280) (96.4 mg,227 μmol, 31.4%) as an orange solid. R_(f)=0.22 (ethyl acetate); HPLCretention time 5.2 min; ¹H NMR (400 MHz, CD₃OD) δ 4.12 (s, 2H),6.65-6.71 (AA′BB′, 2H), 6.88-6.94 (AA′BB′, 2H), 6.94-7.02 (AA′BB′, 2H),7.06-7.12 (AA′BB′, 2H), 7.74-7.84 (AA′BB′, 2H), 7.91-7.98 (AA′BB′, 2H),8.24 (s, 1H); IR (KBr, cm⁻¹) 554, 841, 891, 1132, 1242, 1273, 1341,1437, 1458, 1508, 1543, 1558, 1609, 1653, 2812, 2895, 3096; HRMS (ESI⁺)m/z 426.1458 ([M+H]⁺, C₂₅H₂₀N₃O₄ ⁺ requires 426.1448).

Synthesis Example 2 Coelenteramide (CTMD) analogues modified at the C-3position 2-1) TMD-344 (4a)

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-{5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-phenylpyrazin-2-yl}acetamide (11a)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.07 g, 4.02mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (680 μL, 8.04mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-phenylpyrazin-2-amine (7a)(504 mg, 1.33 mmol) and 4-(dimethylamino)pyridine (16.3 mg, 133 μmol)dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 18hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=4/1) to give Compound 11a (630 mg, 1.01 mmol, 75.4%) as a yellowfoamy solid. R_(f)=0.33 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz,DMSO-d₆) δ 0.18 (s, 6H), 0.23 (s, 6H), 0.94 (s, 9H), 0.96 (s, 9H), 3.47(s, 2H), 6.72-6.79 (AA′BB′, 2H), 6.96-7.02 (AA′BB′, 2H), 7.03-7.09(AA′BB′, 2H), 7.29-7.39 (m, 3H), 7.66-7.74 (m, 2H), 8.05-8.12 (AA′BB′,2H), 8.98 (s, 1H), 10.54 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6(4C), 17.9, 18.0, 25.50 (3C), 25.54 (3C), 41.7, 119.5 (2C), 120.3 (2C),127.8 (2C), 127.9, 128.0 (2C), 128.2 (2C), 128.5, 128.9, 130.3 (2C),130.5, 137.6, 142.8, 147.6, 148.0, 153.8, 156.7, 169.3; IR (KBr, cm⁻¹)523, 694, 781, 804, 839, 914, 1020, 1070, 1086, 1105, 1169, 1263, 1371,1416, 1510, 1605, 1672, 2857, 2886, 2930, 2955, 3040, 3059, 3233; HRMS(ESI⁺) m/z 648.3043 ([M+Na]⁺, C₃₆H₄₇N₃NaO₃Si₂ ⁺ requires 648.3048).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-phenylpyrazin-2-yl]acetamide(4a, TMD-344)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-phenylpyrazin-2-yl]acetamide (11a) (500 mg, 799 mol) in THF (7 mL) was addedtetrabutylammonium fluoride (1.0 M THF solution) (4.00 mL, 4.00 mmol) at0° C., and the mixture was stirred for 40 minutes while elevating toroom temperature. To the mixture was added saturated NH₄Cl aqueoussolution (50 mL) and the product was extracted with ethyl acetate (100mL×3). The combined organic extract was washed successively withsaturated NH₄Cl aqueous solution (200 mL), water (200 mL) and brine (200mL), followed by drying over anhydrous sodium sulfate. After filtrationand concentration under reduced pressure, the residue was recrystallized(n-hexane/ethanol) to give Compound 4a (TMD-344) (206 mg, 517 μmol,64.7%) as a colorless solid. R_(f)=0.41 (dichloromethane/methanol=9/1);HPLC retention time 6.5 min; ¹H NMR (400 MHz, CD₃OD) δ 3.51 (s, 2H),6.69-6.76 (AA′BB′, 2H), 6.87-6.94 (AA′BB′, 2H), 7.02-7.08 (AA′BB′, 2H),7.30-7.43 (m, 3H), 7.58-7.65 (m, 2H), 7.96-8.02 (AA′BB′, 2H), 8.80 (s,1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ 41.8, 115.2 (2C), 116.0 (2C), 125.3,126.5, 128.0 (2C), 128.2 (2C), 128.3 (2C), 128.7, 130.4 (2C), 137.4,137.7, 142.5, 147.8, 148.7, 156.2, 159.3, 169.9; IR (KBr, cm⁻¹) 527,607, 675, 700, 799, 841, 968, 1020, 1165, 1225, 1265, 1323, 1368, 1410,1443, 1458, 1493, 1518, 1537, 1593, 1609, 1676, 3021, 3256, 3368; HRMS(ESI⁺) m/n 420.1321 ([M+Na]⁺, C₂₄H₁₉N₃NaO₃ ⁺ requires 420.1319).

2-2) TMD-343 (4b)

(E)-2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-styrylpyrazin-2-yl]acetamide(11b)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.65 g, 6.19mmol) in CH₂C₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.05 mL, 12.4mmol) at 0° C. and the mixture was stirred for 40 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of(E)-5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-styrylpyrazin-2-amine(7b) (500 mg, 1.24 mmol) and 4-(dimethylamino)pyridine (15.3 mg, 125μmol) dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy) phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 15hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1) to give Compound 11b (563 mg, 864 mol, 69.7%) as a yellowsolid. R_(f)=0.29 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz,DMSO-d₆) δ 0.17 (s, 6H), 0.24 (s, 6H), 0.96 (s, 9H), 0.97 (s, 9H), 3.66(s, 2H), 6.81-6.91 (m, 3H, includes AA′BB′), 6.99-7.04 (AA′BB′, 2H),7.28-7.40 (m, 7H, includes AA′BB′), 7.82 (d, 1H, J=16 Hz), 8.11-8.19(AA′BB′, 2H), 8.91 (s, 1H), 10.66 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ−4.5 (2C), −4.6 (2C), 17.8, 17.9, 25.5 (6C), 42.0, 119.7 (2C), 120.3(2C), 122.5, 127.1 (2C), 128.2 (2C), 128.5, 128.7 (2C), 128.9, 129.0,130.3 (2C), 133.8, 136.0, 137.8, 142.8, 143.9, 148.0, 154.0, 156.7,170.6; IR (KBr, cm⁻¹) 471, 521, 638, 691, 746, 781, 806, 839, 914, 968,1007, 1080, 1103, 1169, 1263, 1325, 1371, 1391, 1414, 1439, 1472, 1510,1566, 1605, 1659, 2857, 2886, 2930, 2055, 3028, 3057, 3217; HRMS (ESI⁺)m/z 674.3203 ([M+Na]⁺, C₃₈H₄₉N₃NaO₃Si₂ ⁺ requires 674.3205).

(E)-2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-styrylpyrazin-2-yl]acetamide(4b, TMD-343)

To a solution of(L)-2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-styrylpyrazin-2-yl]acetamide(11b) (500 mg, 767 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (3.90 mL, 3.90 mmol) at 0° C., and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethanol) to give Compound 4b (TMD-343) (251 mg,592 μmol, 77.3%) as a yellow solid. R_(f)==0.50(dichloromethane/methanol=9/1); HPLC retention time 10.6 min; ¹H NMR(400 MHz, CD₃OD) δ 3.67 (s, 2H), 6.79-6.90 (m, 3H, includes AA′BB),6.90-6.96 (AA′BB′, 2H), 7.25-7.32 (m, 3H, includes AA′BB′), 7.33-7.39(m, 4H), 7.88 (d, 1H, J=16 Hz), 8.00-8.07 (AA′BB′, 2H), 8.72 (s, 1H); 3CNMR (75.5 MHz, DMSO-d₆) δ 42.2, 115.5 (2C), 116.0 (2C), 122.5, 126.2,126.6, 127.3 (2C), 128.4 (2C), 128.8, 128.9 (2C), 130.2 (2C), 133.9,136.1, 137.7, 142.5, 144.1, 148.7, 156.5, 159.3, 171.1; IR (KBr, cm⁻¹)527, 617, 691, 756, 804, 845, 970, 1134, 1175, 1246, 1269, 1327, 1375,1449, 1497, 1512, 1593, 1609, 1638, 3024, 3159, 3566; HRMS (ESI⁺) m/z446.1475 ([M+Na]⁴, C₂₆H₂₁N₃NaO₃ ⁺ requires 446.1475).

2-3) TMD-347 (4c)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(1-phenylvinyl)pyrazin-2-amine(7c)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.00 g, 2.63 mmol) in toluene (30 mL) and ethanol (1.2 mL) weresuccessively added 1-phenylvinylboronic acid (6c) (467 mg, 3.16 mmol),dichlorobis(triphenylphosphine)palladium (II) (110 mg, 157 μmol) and 1 MNa₂CO₃ aqueous solution (2.70 mL, 2.70 mmol) at room temperature, andthe mixture was heated to reflux for 15 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (200mL×3). The combined organic extract was washed successively with water(300 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 100 g,n-hexane/ethyl acetate=3/1) to give Compound 7c (830 mg, 2.06 mmol,78.2%) as a brown oily substance. R_(f)=0.33 (n-hexane/diethylether=2/3); ¹H NMR (400 MHz, DMSO-d₆) δ 0.15 (s, 6H), 0.91 (s, 9H), 5.50(s, 1H), 5.93 (s, 2H), 5.97 (s, 1H), 6.80-6.87 (AA′BB′, 2H), 7.26-7.36(m, 5H), 7.71-7.77 (AA′BB′, 2H), 8.45 (s, 1H); ¹³C NMR (75.5 MHz,DMSO-d₆) δ −4.6 (2C), 17.9, 25.5 (3C), 117.9, 120.0 (2C), 126.3 (2C),126.5 (2C), 128.0, 128.4 (2C), 130.5, 137.7, 138.2, 139.0, 139.2, 144.4,151.8, 154.9; IR (KBr, cm⁻¹) 517, 552, 608, 629, 677, 692, 708, 779,806, 843, 910, 1009, 1026, 1063, 1105, 1148, 1169, 1202, 1263, 1323,1362, 1389, 1423, 1449, 1510, 1605, 2857, 2886, 2928, 2953, 3034, 3055,3175, 3298, 3387, 3481.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-{5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(1-phenylvinyl)pyrazin-2-yl}acetamide(11c)

Under an argon atmosphere, to a mixture of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.80 g, 6.76mmol) in CH₂Cl₂ (15 mL) was added DMF (4 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.20 mL, 14.2mmol) at 0° C. and the mixture was stirred for an hour while elevatingto room temperature. The mixture was concentrated under reduced pressureto give crude 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride(10) as a colorless oil, which was used in the following reactionwithout further purification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(1-phenylvinyl)pyrazin-2-amine(7c) (543 mg, 1.35 mmol) and 4-(dimethylamino)pyridine (15.0 mg, 123μmol) dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C., and the mixture was heated with stirring at 50° C. for16 hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1) to give Compound 11c (292 mg, 448 μmol, 33.3%) as a yellowfoamy solid. R_(f)=0.26 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz,DMSO-d₆) δ 0.13 (s, 6H), 0.18 (s, 6H), 0.91 (s, 9H), 0.92 (s, 9H), 3.22(s, 2H), 5.51 (s, 1H), 5.65 (s, 1H), 6.67-6.74 (AA′BB′, 2H), 6.90-6.97(AA′BB′, 2H), 6.97-7.03 (AA′BB′, 2H), 7.19-7.26 (m, 2H), 7.26-7.31 (m,3H), 7.91-7.98 (AA′BB′, 2H), 8.95 (s, 1H), 10.19 (s, 1H); ¹³C NMR (75.5MHz, acetone-d₆) δ −4.3 (4C), 18.6, 18.7, 26.0 (6C), 42.5, 118.8, 120.5(2C), 121.1 (2C), 127.9 (2C), 128.4, 128.6, 128.78 (2C), 128.84 (2C),130.1, 131.3 (2C), 138.4, 140.0, 144.2, 146.9, 148.7, 148.9, 155.1,157.9, 169.7; IR (KBr, cm⁻¹) 546, 696, 781, 806, 839, 914, 1007, 1082,1169, 1261, 1362, 1420, 1435, 1472, 1508, 1605, 1670, 2857, 2886, 2930,2955, 3030, 3055, 3231 HRMS (ESI⁺) m/z 652.3390 ([M+H]^(,) C₃₈H₅₀N₃O₃Si₂⁺ requires 652.3385).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(1-phenylvinyl)pyrazin-2-yl]acetamide(4c, TMD-347)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(1-phenylvinyl)pyrazin-2-yl]acetamide(11c) (240 mg, 368 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.90 mL, 1.90 mmol) at 0° C. and themixture was stirred for an hour while elevating to room temperature. Tothe mixture was added saturated NH₄Cl aqueous solution (50 mL) and theproduct was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethanol) to give Compound 4c (TMD-347) (83.9mg, 198 μmol, 53.8%) as a brown solid. R_(f)=0.37(dichloromethane/methanol=9/1); HPLC retention time 8.0 min; ¹H NMR (400MHz, DMSO-d₆) δ 3.15 (s, 2H), 5.52 (s, 1H), 5.66 (s, 1H), 6.58-6.64(AA′BB′, 2H), 6.80-6.86 (AA′BB′, 2H), 6.86-6.93 (AA′BB′, 2H), 7.19-7.25(m, 2H), 7.25-7.31 (m, 3H), 7.84-7.91 (AA′BB′, 2H), 8.89 (s, 1H), 9.20(s, 1H), 9.83 (s, 1H), 10.54 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ41.1, 115.0 (2C), 115.9 (2C), 118.1, 125.4, 126.5, 127.1 (2C), 127.7,128.15 (2C), 128.20 (2C), 130.3 (2C), 137.8, 139.0, 143.1, 145.5, 148.1,148.7, 156.1, 159.2, 169.6; IR (KBr, cm⁻¹) 519, 546, 606, 698, 779, 837,916, 1107, 1171, 1231, 1321, 1431, 1481, 1514, 1609, 1670, 3026, 3265;HRMS (ESI⁺) m/z 446.1483 ([M+Na]⁺, C₂₆H₂₁N₃NaO₃+ requires 446.1475).

2-4) TMD-338 (4d)

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(naphthalen-1-yl)pyrazin-2-yl]acetamide (11d)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.00 mL, 11.8mmol) at 0° C. and the mixture was stirred for 40 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(naphthalen-1-yl)pyrazin-2-amine(7d) (500 mg, 1.17 mmol) and 4-(dimethylamino)pyridine (15.3 mg, 125μmol) dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) at 0° C.and the mixture was heated with stirring at 50° C. for 16 hours. Aftercooling to room temperature, to this was added water and the product wasextracted with ethyl acetate (200 mL×3). The combined organic extractwas washed successively with water (200 mL) and brine (200 mL), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by columnchromatography (silica gel 50 g, n-hexane/ethyl acetate=3/1) to giveCompound 11d (442 mg, 653 μmol, 55.9%) as a yellow foamy solid.R_(f)=0.26 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz, DMSO-d₆) δ0.13 (s, 6H), 0.19 (s, 6H), 0.91 (s, 9H), 0.92 (s, 9H), 3.24 (s, 2H),6.54-6.60 (AA′BB′, 2H), 6.65-6.72 (AA′BB′, 2H), 6.91-6.98 (AA′BB′, 2H),7.36-7.54 (m, 4H), 7.61-7.69 (m, 1H), 7.87-8.08 (m, 4H, includesAA′BB′), 9.09 (s, 1H), 10.27 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6(4C), 17.90, 17.94, 25.48 (3C), 25.55 (3C), 119.4 (2C), 120.4 (2C),125.0, 125.4, 125.7, 126.2, 126.8, 128.0, 128.15, 128.21 (2C), 128.6,128.9 (2C), 129.9, 130.7, 133.4, 134.8, 138.2, 144.4, 147.9, 148.3,153.5, 156.7, 169.3 (one carbon at the benzyl position was unobservabledue to overlapping with the septet peak of DMSO); IR (KBr, cm⁻¹) 492,538, 675, 718, 779, 806, 839, 914, 1007, 1080, 1115, 1169, 1260, 1362,1420, 1441, 1472, 1510, 1605, 1670, 2857, 2886, 2930, 2955, 3046, 3223;HRMS (ESI⁺) m/z 698.3205 ([M+Na]⁺, C₄₀H₄₉N₃NaO₃Si₂ ⁺ requires 698.3205).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(naphthalen-1-yl)pyrazin-2-yl]acetamide(4d, TMD-338)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(naphthalen-1-yl)pyrazin-2-yl]acetamide(1 d) (291 mg, 430 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (2.20 mL, 2.20 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with saturated NH₄Cl aqueoussolution (200 mL), water (200 mL) and brine (200 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was recrystallized (n-hexane/ethanol) togive Compound 4d (TMD-338) (181 mg, 404 μmol, 93.8%) as a brown solid.R_(f)=0.56 (dichloromethane/methanol=9/1); HPLC retention time 9.1 min;¹H NMR (400 MHz, DMSO-d₆) δ 3.17 (s, 2H), 6.45-6.53 (AA′BB′, 2H),6.56-6.64 (AA′BB′, 2H), 6.80-6.88 (AA′BB′, 2H), 7.35-7.56 (m, 4H),7.61-7.68 (m, 1H), 7.88-8.04 (m, 4H, includes AA′BB′), 9.03 (s, 1H),9.15 (s, 1H), 9.85 (s, 1H), 10.14 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ41.2, 115.0 (2C), 115.9 (2C), 125.1, 125.3, 125.4, 125.8, 126.3, 126.5,126.9, 128.25, 128.27 (2C), 128.7, 129.8 (2C), 130.7, 133.4, 134.9,138.0, 144.0, 148.38, 148.44, 155.9, 159.2, 169.8; IR (KBr, cm⁻¹) 492,540, 623, 777, 802, 839, 982, 1078, 1171, 1231, 1317, 1364, 1447, 1477,1516, 1609, 1670, 3055, 3246; HRMS (ESI⁾ m/z 470.1483 ([M+Na]⁺,C₂₈H₂₁N₃NaO₃ ⁺ requires 470.1475).

2-5) TMD-339 (4e)

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(naphthalen-2-yl)pyrazin-2-yl]acetamide(11e)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.00 mL, 11.8mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(naphthalen-2-yl)pyrazin-2-amine(7e) (500 mg, 1.17 mmol) and 4-(dimethylamino)pyridine (15.0 mg, 123μmol) dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C., and the mixture was heated with stirring at 50° C. for16 hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1) to give Compound 11e (460 mg, 681 μmol, 58.2%) as a yellowfoamy solid. R_(f)=0.26 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz,DMSO-d₆) δ 0.12 (s, 6H), 0.21 (s, 6H), 0.91 (s, 9H), 0.94 (s, 9H), 3.46(s, 2H), 6.55-6.65 (AA′BB′, 2H), 6.91-6.97 (AA′BB′, 2H), 6.97-7.03(AA′BB′, 2H), 7.47-7.58 (m, 2H), 7.75-8.00 (m, 4H), 8.08-8.14 (AA′BB′,2H), 8.23 (s, 1H), 9.00 (s, 1H), 10.63 (s, 1H); ¹³C NMR (75.5 MHz,DMSO-d₆) δ −4.6 (4C), 17.8, 17.9, 25.5 (6C), 119.4 (2C), 120.3 (2C),125.5, 126.1, 126.6, 127.1, 127.2, 127.3, 127.5, 127.9, 128.2 (2C),128.5, 130.2 (2C), 132.6, 132.8, 135.3, 137.6, 143.0, 147.5, 148.0,153.7, 156.7, 169.4 (one carbon at the benzyl position was unobservabledue to overlapping with the septet peak of DMSO); IR (KBr, cm⁻¹) 478,527, 687, 718, 746, 781, 804, 839, 914, 1009, 1082, 1103, 1128, 1169,1261, 1362, 1415, 1445, 1472, 1508, 1605, 1670, 2857, 2886, 2928, 2955,3057, 3221; HRMS (ESI⁺) m/z 698.3194 ([M+Na]+, C₄₀H₄₉N₃NaO₃Si₂ ⁺requires 698.3205).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(naphthalen-2-yl)pyrazin-2-yl]acetamide(4e, TMD-339)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}]-3-(naphthalen-2-yl)pyrazin-2-yl]acetamide(11e) (300 mg, 444 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (2.30 mL, 2.30 mmol) at 0° C., and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with saturated NH₄Cl aqueoussolution (200 mL), water (200 mL) and brine (200 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was recrystallized (n-hexane/ethanol) togive Compound 4e (TMD-339) (131 mg, 292 μmol, 65.8%) as a brown solid.R_(f)=0.48 (dichloromethane/methanol=9/1); HPLC retention time 10.9 min;¹H NMR (400 MHz, DMSO-d₆) δ 3.39 (s, 2H), 6.50-6.58 (AA′BB′, 2H),6.82-6.94 (2AA′BB′, 4H), 7.46-7.58 (m, 2H), 7.79-7.91 (m, 4H), 8.02-8.08(AA′BB′, 2H), 8.23 (s, 1H), 8.94 (s, 1H), 9.20 (s, 1H), 9.88 (s, 1H),10.52 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ 41.6, 115.1 (2C), 115.9(2C), 125.3, 125.6, 126.3, 126.5, 126.8, 127.3, 127.5, 127.6, 128.3(2C), 128.6, 130.2 (2C), 132.7, 132.9, 135.3, 137.3, 142.7, 147.5,148.6, 156.1, 159.3, 169.8; IR (KBr, cm⁻¹) 529, 635, 669, 752, 826, 968,1163, 1223, 1356, 1454, 1491, 1516, 1539, 1593, 1611, 1672, 3065, 3246;HRMS (ESI⁺) m/z 470.1486 ([M+Na]⁺, C₂₈H₂₁N₃NaO₃ ⁺ requires 470.1475).

2-6) TMD-340 (4f)

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(thiophen-2-yl)pyrazin-2-yl]acetamide(11f)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (2.12 g, 7.96mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.35 mL, 16.0mmol) at 0° C. and the mixture was stirred for an hour while elevatingto room temperature. The mixture was concentrated under reduced pressureto give crude 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride(10) as a colorless oil, which was used in the following reactionwithout further purification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thiophen-2-yl)pyrazin-2-amine(7f) (600 mg, 1.59 mmol) and 4-(dimethylamino)pyridine (19.8 mg, 162μmol) dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 15hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1). The resulting solid was recrystallized (n-hexane/ethylacetate) to give Compound 11f (446 mg, 706 μmol, 44.4%) as a colorlesssolid. R_(f)=0.26 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz,DMSO-d₆) δ 0.18 (s, 6H), 0.23 (s, 6H), 0.94 (s, 9H), 0.97 (s, 9H), 3.65(s, 2H), 0.678-6.86 (AA′BB′, 2H), 6.97-7.06 (m, 3H, includes AA′BB′),7.19-7.27 (AA′BB′, 2H), 7.51 (dd, 1H, J=0.9, 3.7 Hz), 7.69 (dd, 1H,J=0.9, 5.0 Hz), 8.06-8.14 (AA′BB′, 2H), 8.93 (s, 1H), 10.60 (s, 1H); ¹³CNMR (75.5 MHz, DMSO-d₆) δ −4.6 (4C), 17.9, 18.0, 25.50 (3C), 25.54 (3C),41.9, 119.6 (2C), 120.4 (2C), 127.8, 127.9, 128.0, 128.2 (2C), 128.4,129.5, 130.5 (2C), 137.3, 140.5, 140.6, 142.5, 148.0, 153.9, 157.0,170.3; IR (KBr, cm⁻¹) 419, 519, 673, 704, 741, 781, 802, 839, 916, 1169,1265, 1335, 1362, 1373, 1414, 1439, 1472, 1510, 1605, 1667, 2857, 2887,2930, 2955, 3221; HRMS (ESI⁺) m/z 654.2617 ([M+Na]⁺, C₃₄H₄₅N₃NaO₃Si₂ ⁺requires 654.2612).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(thiophen-2-yl)pyrazin-2-yl]acetamide(4f, TMD-340)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(thiophen-2-yl)pyrazin-2-yl]acetamide (11f) (252 mg, 398 μmol) in THF (4 mL)added tetrabutylammonium fluoride (1.0 M THF solution) (2.00 mL, 2.00mmol) at 0° C., and the mixture was stirred for an hour while elevatingto room temperature, To the mixture was added saturated NH₄Cl aqueoussolution (50 mL) and the product was extracted with ethyl acetate (100mL×3). The combined organic extract was washed successively withsaturated NH₄Cl aqueous solution (200 mL), water (200 mL) and brine (200mL), followed by drying over anhydrous sodium sulfate. Filtration andconcentration under reduced pressure gave Compound 4f (TMD-340) (160 mg,397 μmol, 99.6%) as a colorless solid. R_(f)=0.37(dichloromethane/methanol=9/1); HPLC retention time 6.8 min; ¹H NMR (400MHz, DMSO-d₆) δ 3.59 (s, 2H), 6.70-6.76 (AA′BB′, 2H), 6.90-6.96 (AA′BB′,2H), 6.99 (dd, 1H, J=3.8, 5.0 Hz), 7.12-7.18 (AA′BB′, 2H), 7.48 (dd, 1H,J=1.1, 3.8 Hz), 7.69 (dd, 1H, J=1.1, 5.0 Hz), 8.01-8.07 (AA′BB′, 2H),8.89 (s, 1H), 9.32 (s, 1H), 9.97 (s, 1H), 10.50 (s, 1H), ¹³C NMR (75.5MHz, DMSO-d₆) δ 42.0, 115.2 (2C), 116.0 (2C), 125.2, 126.0, 128.1,128.25, 128.34 (2C), 129.6, 130.5 (2C), 137.1, 140.1, 140.8, 142.7,148.6, 156.3, 159.5, 170.8; IR (KBr, cm⁻¹) 523, 631, 698, 725, 791, 841,966, 984, 1074, 1113, 1167, 1227, 1263, 1317, 1375, 1406, 1431, 1450,1495, 1518, 1535, 1593, 1609, 1674, 3021, 3244, 3372; HRMS (ESI⁺) m/z426.0877 ([M+Na]⁴, C₂₂H₁₇N₃NaO₃S⁺ requires 426.0883).

2-7) TMD-345 (4g)

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-{5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thiophen-3-yl)pyrazin-2-yl}acetamide(11g)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.00 mL, 11.8mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thiophen-3-yl)pyrazin-2-amine(7g) (450 mg, 1.17 mmol) and 4-(dimethylamino)pyridine (15.5 mg, 127μmol) dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 15hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1) to give Compound 1 g (512 mg, 809 μmol, 69.0%) as a yellowsolid. R_(f)=0.22 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz,DMSO-d₆) δ 0.16 (s, 6H), 0.20 (s, 6H), 0.92 (s, 9H), 0.94 (s, 9H), 3.56(s, 2H), 6.72-6.82 (AA′BB′, 2H), 6.93-7.02 (AA′BB′, 2H), 7.12-7.22(AA′BB′, 2H), 7.50 (dd, 1H, J=2.9, 5.0 Hz), 7.59 (d, 1H, J=5.0 Hz), 7.81(d, 1H, J=2.9 Hz), 8.04-8.12 (AA′BB′, 2H), 8.91 (s, 1H), 10.50 (s, 1H);¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (4C), 17.90, 17.95, 25.50 (3C), 25.54(3C), 41.9, 119.6 (2C), 120.3 (2C), 125.7, 126.0, 127.7, 127.9, 128.2(2C), 128.8, 130.5 (2C), 137.1, 138.2, 142.0, 143.5, 148.0, 153.9,156.8, 169.9; IR (KBr, cm⁻¹) 517, 669, 712, 741, 781, 806, 839, 914,1007, 1082, 1105, 1169, 1261, 1341, 1362, 1441, 1472, 1508, 1605, 1665,2857, 2886, 2930, 2955, 3057, 3227; HRMS (ESI⁺) m/z 654.2618 ([M+Na]⁺,C₃₄H₄₅N₃NaO₃Si₂ ⁺ requires 654.2612).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(thiophen-3-yl)pyrazin-2-yl]acetamide(4g, TMD-345)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(thiophen-3-yl)pyrazin-2-yl]acetamide (11g) (310 mg, 491 μmol) in THF (5 mL)was added tetrabutylammonium fluoride (1.0 M THF solution) (2.50 mL,2.50 mmol) at 0° C. and the mixture was stirred for an hour whileelevating to room temperature. To the mixture was added saturated NH₄Claqueous solution (50 mL) and the product was extracted with ethylacetate (100 mL×3). The combined organic extract was washed successivelywith water (200 mL) and brine (200 mL), followed by drying overanhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was recrystallized (n-hexane/ethanol) togive Compound 4g (TMD-345) (85.0 mg, 211 μmol, 43.0%) as a yellow solid.R_(f)=0.50 (dichloromethane/methanol=9/1); HPLC retention time 6.2 min;¹H NMR (400 MHz, DMSO-d₆) δ 3.50 (s, 2H), 6.65-6.72 (AA′BB′, 2H),6.84-6.92 (AA′BB′, 2H), 7.03-7.11 (AA′BB′, 2H), 7.49 (dd, 1H, J=2.9, 5.0Hz), 7.58 (dd, 1H, J=1.2, 5.0 Hz), 7.80 (dd, 1H, J=1.2, 2.9 Hz),7.98-8.06 (AA′BB′, 2H), 8.85 (s, 1H), 9.27 (s, 1H), 9.87 (s, 1H), 10.39(s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ 41.9, 115.2 (2C), 115.9 (2C),125.2, 125.9, 126.2, 126.4, 127.8, 128.3 (2C), 130.4 (2C), 136.9, 138.3,141.6, 143.7, 148.6, 156.3, 159.3, 170.4; IR (KBr, cm⁻¹) 419, 517, 609,671, 706, 783, 804, 839, 934, 1040, 1082, 1111, 1173, 1227, 1248, 1272,1312, 1352, 1441, 1516, 1593, 1611, 1674, 2808, 3250; HRMS (ESI⁺) m/z426.0878 ([M+Na]⁺, C₂₂H₁₇N₃NaO₃S⁺ requires 426.0883).

2-8) TMD-342 (4h)

N-[3-(Benzo[b]thiophen-2-yl)-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11h)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (985 μL, 11.6mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of3-(benzo[b]thiophen-2-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7h) (505 mg, 1.16 mmol) and 4-(dimethylamino)pyridine (15.0 mg, 123μmol) dissolved in anhydrous pyridine (20 mL) was added 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) prepared above at 0° C. andthe mixture was heated with stirring at 50° C. for 15 hours. Aftercooling to room temperature, to this was added water and the product wasextracted with ethyl acetate (200 mL×3). The combined organic extractwas washed successively with water (200 mL) and brine (200 mL), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by columnchromatography (silica gel 50 g, n-hexane/ethyl acetate=3/1). Theresulting solid was recrystallized (n-hexane/ethanol) to give Compound11h (294 mg, 431 μmol, 37.0%) as a colorless solid. R_(f)=0.19(n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz, DMSO-d₆) δ 0.19 (s, 6H),0.25 (s, 6H), 0.96 (s, 9H), 0.97 (s, 9H), 3.71 (s, 2H), 6.82-6.89(AA′BB′, 2H), 7.01-7.08 (AA′BB′, 2H), 7.26-7.42 (m, 4H, includesAA′BB′), 7.60-7.65 (m, 2H), 7.96 (d, 1H, J=7.9 Hz), 8.10-8.18 (AA′BB′,2H), 9.02 (s, 1H), 10.82 (s, 1H), IR (KBr, cm⁻¹) 525, 677, 692, 710,727, 743, 779, 797, 808, 835, 922, 972, 1011, 1074, 1171, 1256, 1327,1362, 1391, 1416, 1445, 1503, 1603, 1657, 859, 2886, 2930, 2957, 3059,3161, 3211; HRMS (ESI⁺) m/z 704.2758 ([M+Na]⁺, C₃₈H₄₇N₃NaO₃SSi₂ ⁺requires 704.2769).

N-[3-(Benzo[b]thiophen-2-yl)-5-(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)acetamide (4h, TMD-342)

To a solution ofN-[3-(benzo[b]thiophen-2-yl)-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11h) (186 mg, 273 μmol) in THF (2 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.40 mL, 1.40 mmol) at 0° C. and themixture was stirred for an hour while elevating to room temperature. Tothe mixture was added saturated NH₄Cl aqueous solution (50 mL) and theproduct was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with saturated NH₄Cl aqueoussolution (200 mL), water (200 mL) and brine (200 mL), followed by dryingover anhydrous sodium sulfate. Filtration and concentration underreduced pressure gave Compound 4h (TMD-342) (123 mg, 271 μmol, 99.3%) asa colorless solid. R_(f)=0.41 (dichloromethane/methanol=9/1); HPLCretention time 13.1 min; ¹H NMR (400 MHz, DMSO-d₆) δ 3.62 (s, 2H),6.70-6.78 (AA′BB′, 2H), 6.89-6.95 (AA′BB′, 2H), 7.17-7.22 (AA′BB′, 2H),7.30-7.39 (m, 2H), 7.51-7.59 (m, 2H), 7.90-7.95 (m, 1H), 8.01-8.09(AA′BB′, 2H), 8.94 (s, 1H), 9.38 (s, 1H), 9.96 (s, 1H), 10.70 (s, 1H);¹³C NMR (75.5 MHz, DMSO-d₆) 842.3, 115.4 (2C), 116.1 (2C), 122.3, 124.3,124.60, 124.63, 125.1, 125.6, 125.8, 128.4 (2C), 130.5 (2C), 137.8,139.9, 140.3, 141.0, 141.5, 142.0, 148.5, 156.5, 159.6, 170.6; IR (KBr,cm⁻¹) 525, 554, 579, 623, 654, 681, 727, 804, 827, 847, 972, 1013, 1070,1128, 1175, 1242, 1314, 1377, 1439, 1497, 1514, 1593, 1676, 3302; HRMS(ESI⁺) m/z 476.1052 ([M+Na]⁺, C₂₆H₁₉N₃NaO₃S⁺ requires 476.1039).

2-9) TMD-346 (4i)

N-[3-(Benzo[b]thiophen-3-yl)-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11i)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.00 mL, 11.8mmol) at 0° C. and the mixture was stirred for an hour while elevatingto room temperature. The mixture was concentrated under reduced pressureto give crude 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride(10) as a colorless oil, which was used in the following reactionwithout further purification.

Under an argon atmosphere, to a mixture of3-(benzo[b]thiophen-3-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7i) (505 mg, 1.16 mmol) and 4-(dimethylamino)pyridine (15.0 mg, 123μmol) dissolved in anhydrous pyridine (20 mL) was added 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) prepared above at 0° C. andthe mixture was heated with stirring at 50° C. for 15 hours. Aftercooling to room temperature, to this was added water and the product wasextracted with ethyl acetate (200 mL×3). The combined organic extractwas washed successively with water (200 mL) and brine (200 mL), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by columnchromatography (silica gel 50 g, n-hexane/ethyl acetate=3/1) to giveCompound 11i (505 mg, 740 μmol, 63.5%) as a yellow solid. R_(f)=0.19(n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz, DMSO-d₆) δ 0.15 (s, 6H),0.20 (s, 6H), 0.91 (s, 91-H), 0.93 (s, 9H), 3.41 (s, 2H), 6.65-6.71(AA′BB′, 2H), 6.93-7.00 (2AA′BB′, 4H), 7.35-7.42 (m, 2H), 7.80 (s, 1H),7.95-8.07 (m, 4H, includes AA′BB′), 9.01 (s, 1H), 10.49 (s, 1H); ¹³C NMR(75.5 MHz, DMSO-d₆) δ −4.5 (4C), 17.89, 17.93, 25.47 (3C), 25.54 (3C),41.6, 119.5 (2C), 120.4 (2C), 122.6, 123.5, 124.4 (2C), 127.3, 127.9,128.1 (2C), 128.9, 130.3 (2C), 132.3, 137.5, 137.5, 139.4, 143.7, 143.8,147.6, 153.8, 156.7, 169.4; IR (KBr, cm⁻¹) 683, 700, 735, 758, 781, 804,839, 914, 1169, 1261, 1331, 1362, 1435, 1472, 1508, 1605, 1670, 2857,2886, 2930, 2955; HRMS (ESI⁺) m/z 704.2781 ([M+Na]⁺, C₃₈H₄₇N₃NaO₃SSi₂ ⁺requires 704.2769).

N-[3-(Benzo[b]thiophen-3-yl)-5-(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)acetamide (4i, TMD-346)

To a solution ofN-[3-(benzo[b]thiophen-3-yl)-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11i) (300 mg, 440 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (2.20 mL, 2.20 mmol) at 0° C. and themixture was stirred for an hour while elevating to room temperature. Tothe mixture was added saturated NH₄Cl aqueous solution (50 mL) and theproduct was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), and dried over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was recrystallized(n-hexane/ethanol) to give Compound 4i (TMD-346) (191 mg, 422 μmol,95.8%) as a brown solid. R_(f)=0.50 (dichloromethane/methanol=9/1); HPLCretention time 9.6 min; ¹H NMR (400 MHz, DMSO-d₆) δ 3.36 (s, 2H),6.57-6.63 (AA′BB′, 2H), 6.84-6.92 (2AA′BB′, 4H), 7.36-7.43 (m, 2H), δ 79(s, 1H), 7.95-8.04 (m, 4H, includes AA′BB′), 8.95 (s, 1H), 9.22 (s, 1H),9.88 (s, 1H), 10.37 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ 41.7, 115.1(2C), 116.0 (2C), 122.8, 123.6, 124.5 (2C), 125.2, 126.5, 127.5, 128.2(2C), 130.2 (2C), 132.4, 137.2, 137.6, 139.5, 143.3, 144.0, 148.2,156.1, 159.3, 169.9; IR (KBr, cm⁻¹) 419, 473, 525, 611, 735, 760, 837,970, 1171, 1233, 1329, 1435, 1479, 1514, 1609, 1668, 2957, 3260; HRMS(ESI⁺) m/z 476.1052 ([M+Na]⁺, C₂₆H₁₉N₃O₃S⁺ requires 476.1039).

2-10) TMD-341 (4j)

N-[3,5-Bis{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11j)

Under an argon atmosphere, to a mixture of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (985 μL, 11.6mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of3,5-bis[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (7j) (591mg, 1.16 mmol) and 4-(dimethylamino)pyridine (15.7 mg, 129 mol)dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 15hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=4/1) to give Compound 11j (714 mg, 945 μmol, 81.1%) as a yellowfoamy solid. R_(f)=0.37 (n-hexane/diethyl ether=2/3); ¹H NMR (400 MHz,DMSO-d₆) δ 0.14 (s, 6H), 0.19 (s, 6H), 0.20 (s, 6H), 0.92 (s, 9H), 0.95(s, 18H), 3.47 (s, 2H), 6.71-6.78 (2AA′BB′, 4H), 6.94-7.02 (AA′BB′, 2H),7.03-7.13 (AA′BB′, 2H), 7.57-7.63 (AA′BB′, 2H), 8.03-8.10 (AA′BB′, 2H),8.91 (s, 1H), 10.49 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.5 (6C),17.9 (3C), 25.5 (9C), 41.7, 119.3 (2C), 119.5 (2C), 120.3 (2C), 127.9,128.1 (2C), 128.9, 129.3 (2C), 130.4 (2C), 130.7, 136.9, 142.4, 147.2,147.8, 153.8, 155.7, 156.7, 169.3; IR (KBr, cm⁻¹) 523, 637, 677, 710,781, 804, 839, 914, 1009, 1078, 1105, 1169, 1258, 1362, 1402, 1416,1441, 1472, 1510, 1605, 1667, 2857, 2886, 2928, 2955, 3036, 3221; HRMS(ESP) m/z 778.3853 ([M+Na]⁺, C₄₂H₆₁N₃NaO₄Si₃ ⁺ requires 778.3862).

N-[3, 5-Bis(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)acetamide(4j, TMD-341)

To a solution ofN-[3,5-bis{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11j) (500 mg, 661 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (5.00 mL, 5.00 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethanol) to give Compound 4j (TMD-341) (263 mg,637 μmol, 96.3%) as a brown solid. R_(f)=0.43(dichloromethane/methanol=9/1); HPLC retention time 4.3 min; ¹H NMR (400MHz, DMSO-d₆) δ 3.40 (s, 2H), 6.60-6.68 (AA′BB′, 2H), 6.68-6.74 (AA′BB′,2H), 6.82-6.90 (AA′BB′, 2H), 6.94-7.03 (AA′BB′, 2H), 7.54-7.62 (AA′BB′,2H), 7.94-8.02 (AA′BB′, 2H), 8.80 (s, 1H), 9.24 (s, 1H), 9.70 (s, 1H),9.85 (s, 1H), 10.29 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ 41.7, 115.1(4C), 115.9 (2C), 125.4, 126.6, 128.1, 128.2 (2C), 129.6 (2C), 130.3(2C), 136.3, 141.9, 147.8, 148.4, 156.1, 158.3, 159.2, 170.0; IR (KBr,cm⁻¹) 530, 571, 839, 966, 1011, 1080, 1171, 1234, 1321, 1369, 1420,1481, 1516, 1609, 1670, 3246; HRMS (ESI⁺) m/z 436.1269 ([M+Na]⁺,C₂₄H₁₉N₃NaO₄ ⁺ requires 436.1268).

2-11) TMD-373 (4k)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(thieno[3,2-b]thiophen-2-yl)pyrazin-2-amine(7k)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (500mg, 1.31 mmol) in toluene (15 mL) and ethanol (600 μL) were successivelyadded thieno[3,2-b]thiophen-2-boronic acid (6k) (290 mg, 1.58 mmol),dichlorobis(triphenylphosphine)palladium (II) (56.0 mg, 79.8 μmol) and 1M Na₂CO₃ aqueous solution (1.40 mL, 1.40 mmol) at room temperature andthe mixture was heated to reflux for 21 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (100mL×3). The combined organic extract was washed successively with water(200 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 50 g,n-hexane/ethyl acetate=3/1) to give Compound 7k (278 mg, 632 μmol,48.0%) as an orange solid. R_(f)=0.19 (n-hexane/ethyl acetate=3/1); ¹HNMR (400 MHz, DMSO-d₆) δ 0.19 (s, 6H), 0.94 (s, 9H), 6.55 (s, 2H),6.89-6.96 (AA′BB′, 2H), 7.44 (d, 1H, J=5.3 Hz), 7.71 (d, 1H, J=5.3 Hz),7.86-7.94 (AA′BB′, 2H), 8.08 (s, 1H), 8.49 (s, 1H); IR (KBr, cm⁻) 509,637, 702, 781, 839, 916, 986, 1103, 1165, 1261, 1344, 1418, 1464, 1510,1605, 1638, 2857, 2930, 2953, 3156, 3296, 3416.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(thieno[3,2-b]thiophen-2-yl)pyrazin-2-yl]acetamide(11k)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (757 mg, 2.84mmol) in CH₂Cl₂ (8 mL) was added DMF (2 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (490 μL, 5.79mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thieno[3,2-b]thiophen-2-yl)pyrazin-2-amine(7k) (250 mg, 569 μmol) and 4-(dimethylamino)pyridine (12.3 mg, 101μmol) dissolved in anhydrous pyridine (15 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 13hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 30 g, n-hexane/ethylacetate=3/1) to give Compound 1 k (228 mg, 331 μmol, 58.2%) as a yellowsolid. R=0.26 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz, DMSO-d₆) δ0.17 (s, 6H), 0.22 (s, 6H), 0.93 (s, 9H), 0.95 (s, 9H), 3.67 (s, 2H),6.78-6.86 (AA′BB′, 2H), 6.96-7.04 (AA′BB′, 2H), 7.20-7.30 (AA′BB′, 2H),7.41 (d, 1H, J=5.2 Hz), 7.57 (s, 1H), 7.73 (d, 1H, J=5.2 Hz), 8.04-8.13(AA′BB′, 2H), 8.92 (s, 1H), 10.69 (s, 1H); IR (KBr, cm⁻¹) 527, 635, 702,779, 837, 918, 1007, 1074, 1169, 1256, 1362, 1445, 1508, 1603, 1655,2857, 2886, 2928, 2955, 3211.

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(thieno[3,2-b]thiophen-2-yl)pyrazin-2-yl]acetamide(4k, TMD-373)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-(4-(tert-butyldimethylsilyloxy)phenyl)-3-(thieno[3,2-b]thiophen-2-yl)pyrazin-2-yl]acetamide(11k) (172 mg, 250 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.30 mL, 1.30 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4k (TMD-373)(88.4 mg, 192 μmol, 77.1%) as a yellow solid. R_(f)=0.37(dichloromethane/methanol=9/1); HPLC retention time 10.6 min; ¹H NMR(400 MHz, DMSO-d₆) δ 3.59 (s, 2H), 6.71-6.78 (AA′BB′, 2H), 6.85-6.94(AA′BB′, 2H), 7.15-7.23 (AA′BB′, 2H), 7.37-7.42 (m, 2H), 7.72 (d, 1H,J=5.2 Hz), 7.99-8.05 (AA′BB′, 2H), 8.87 (s, 1H), 9.38 (s, 1H), 9.95 (s,1H), 10.61 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ 42.3, 115.4 (2C),115.9 (2C), 120.1, 120.5, 125.0, 125.8, 128.3 (2C), 130.4 (2C), 130.5,137.1, 139.7, 140.0, 141.0, 142.4, 142.9, 148.4, 156.5, 159.5, 170.5; IR(KBr, cm⁻¹) 530, 635, 704, 725, 804, 986, 1173, 1242, 1325, 1371, 1439,1491, 1514, 1609, 1676, 3304.

2-12) TMD-374 (4l)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(dithieno[3,2-b:2′,3′-d]thiophen-2-yl)pyrazin-2-amine(7l)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (700mg, 1.84 mmol) in toluene (21 mL) and ethanol (840 μL) were successivelyadded dithieno[3,2-b:2′,3′-d]thiophen-2-boronic acid (61) (540 mg, 2.25mmol), dichlorobis(triphenylphosphine)palladium (II) (78.2 mg, 111 μmol)and 1 M Na₂CO₃ aqueous solution (1.90 mL, 1.90 mmol) at roomtemperature, and the mixture was heated to reflux for 18 hours. Aftercooling to room temperature, to the mixture was added water and themetal catalyst was removed by filtration. The product was extracted withethyl acetate (100 mL×3). The combined organic extract was washedsuccessively with water (300 mL) and brine (300 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 100 g, n-hexane/ethyl acetate=3/1) to give Compound 71 (477mg, 963 μmol, 52.3%) as a yellow solid. R_(f)=0.26 (n-hexane/ethylacetate=3/1); ¹H NMR (400 MHz, DMSO-d₆) δ 0.20 (s, 6H), 0.94 (s, 9H),6.59 (s, 2H), 6.89-6.97 (AA′BB′, 2H), 7.53 (d, 1H, J=5.2 Hz), 7.74 (d,1H, J=5.2 Hz), 7.88-7.97 (AA′BB′, 2H), 8.16 (s, 1H), 8.51 (s, 1H); IR(KBr, cm⁻¹) 530, 625, 644, 698, 781, 839, 908, 1072, 1109, 1179, 1225,1267, 1360, 1393, 1462, 1512, 1605, 2857, 2928, 2957, 3173, 3285, 3424.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(dithieno[3,2-b:2′,3′-d]thiophen-2-yl)pyrazin-2-yl]acetamide(11l)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.00 g, 3.76mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (640 μL, 7.56mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(thieno[3,2-b:2′,3′-d]thiophen-2-yl)pyrazin-2-amine(7l) (370 mg, 746 μmol) and 4-(dimethylamino)pyridine (14.8 mg, 121μmol) dissolved in anhydrous pyridine (15 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 20hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1). The resulting solid was recrystallized (n-hexane/ethylacetate) to give Compound 11l (116 mg, 156 μmol, 20.9%) as a brownsolid. R_(f)=0.19 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz,DMSO-d₆) δ 0.16 (s, 6H), 0.21 (s, 6H), 0.93 (s, 9H), 0.94 (s, 9H), 3.68(s, 2H), 6.78-6.88 (AA′BB′, 2H), 6.94-7.06 (AA′BB′, 2H), 7.20-7.30(AA′BB′, 2H), 7.47 (d, 1H, J=5.2 Hz), 7.65 (s, 1H), 7.75 (d, 1H, J=5.2Hz), 8.06-8.14 (AA′BB′, 2H), 8.93 (s, 1H), 10.72 (s, 1H); IR (KBr, cm⁻¹)530, 602, 644, 700, 779, 837, 922, 1072, 1171, 1265, 1361, 1447, 1504,1603, 1657, 2857, 2928, 2955, 3208.

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(thieno[3,2-b:2′,3′-d]thiophen-2-yl)pyrazin-2-yl]acetamide (4l, TMD-374)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(dithieno[3,2-b:2′,3′-d]thiophen-2-yl)pyrazin-2-yl]acetamide(11l) (82.3 mg, 111 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (600 μL, 600 μmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4l (TMD-374)(50.7 mg, 192 μmol, 88.8%) as a yellow solid. R_(f)=0.37(dichloromethane/methanol=9/1); HPLC retention time 17.7 min; ¹H NMR(400 MHz, DMSO-d₆) δ 3.61 (s, 2H), 6.70-6.80 (AA′BB′, 2H), 6.87-6.94(AA′BB′, 2H), 7.15-7.25 (AA′BB′, 2H), 7.44 (s, 1H), 7.52 (d, 1H, J=5.2Hz), 7.75 (d, 1H, J=5.2 Hz), 8.01-8.09 (AA′BB′, 2H), 8.89 (s, 1H), 9.34(s, 1H), 9.96 (s, 1H), 10.66 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ42.3, 115.4 (2C), 115.9 (2C), 121.4, 121.9, 125.0, 125.7, 128.3 (2C),128.9, 130.3, 130.4 (2C), 132.3, 137.2, 140.0, 141.8, 142.00, 142.02,142.8, 148.4, 156.5, 159.5, 170.5; IR (KBr, cm⁻¹) 534, 598, 644, 706,793, 835, 893, 972, 1109, 1173, 1190, 1242, 1319, 1389, 1439, 1491,1611, 1665, 3231.

2-13) TMD-375 (4m)

3-(2,2′-Bithiophen-5-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7m)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (500mg, 1.31 mmol) in toluene (15 mL) and ethanol (600 μL) were successivelyadded 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,2′-bithiophene(6m) (460 mg, 1.57 mmol), dichlorobis(triphenylphosphine)palladium (II)(56.3 mg, 80.2 μmol) and 1 M Na₂CO₃ aqueous solution (1.40 mL, 1.40mmol) at room temperature, followed by heating to reflux for 15 hoursAfter cooling to room temperature, to the mixture was added water andthe metal catalyst was removed by filtration. The product was extractedwith ethyl acetate (100 mL×3). The combined organic extract was washedsuccessively with water (200 mL) and brine (300 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 50 g, n-hexane/ethyl acetate=3/1) to give Compound 7m (574mg, 1.23 mmol, 93.7%) as a yellow solid. R_(f)=0.19 (n-hexane/ethylacetate=3/1); ¹H NMR (400 MHz, DMSO-d₆) δ 0.19 (s, 6H), 0.94 (s, 9H),6.49 (s, 2H), 6.88-6.97 (AA′BB′, 2H), 7.10 (dd, 1H, J=3.7, 3.7 Hz), 7.34(d, 1H, J=3.7 Hz), 7.41 (d, 1H, J=3.7 Hz), 7.53 (d, 1H, J=4.0 Hz), 7.70(d, 1H, J=4.0 Hz), 7.84-7.92 (AA′BB′, 2H), 8.47 (s, 1H); ¹³C NMR (67.8MHz, DMSO-d₆) δ −4.5 (2C), 18.0, 25.5 (3C), 120.2 (2C), 124.5, 124.9,125.9, 126.5 (2C), 126.6, 128.5, 129.8, 131.2, 136.4, 137.1, 138.1,139.5, 141.6, 149.8, 155.3; IR (KBr, cm⁻¹) 505, 544, 694, 781, 806, 839,914, 980, 1011, 1084, 1169, 1209, 1258, 1375, 1418, 1454, 1512, 1605,2857, 2928, 2953, 3177, 3296, 3453.

N-[3-(2,2′-Bithiophen-5-yl)-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11m)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.00 g, 3.76mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (640 μL, 7.56mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of3-(2,2′-bithiophen-5-yl)-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine(7m) (350 mg, 752 μmol) and 4-(dimethylamino)pyridine (15.0 mg, 123μmol) dissolved in anhydrous pyridine (15 mL) was added 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) prepared above at 0° C. andthe mixture was heated with stirring at 50° C. for 20 hours. Aftercooling to room temperature, to this was added water and the product wasextracted with ethyl acetate (100 mL×3). The combined organic extractwas washed successively with water (200 mL) and brine (200 mL), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by columnchromatography (silica gel 50 g, n-hexane/ethyl acetate=3/1) to giveCompound 11m (298 mg, crude) as a brown oily substance. R_(f)=0.26(n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz, DMSO-d₆) δ 0.16 (s, 6H),0.22 (s, 6H), 0.92 (s, 9H), 0.94 (s, 9H), 3.64 (s, 2H), 6.76-6.85(AA′BB′, 2H), 6.98-7.05 (AA′BB′, 2H), 7.06-7.13 (m, 2H), 7.19-7.27(AA′BB′, 2H), 7.33 (d, 1H, J=3.6 Hz), 7.36 (d, 1H, J=3.6 Hz), 7.54-7.59(m, 1H), 8.05-8.11 (AA′BB′, 2H), 8.91 (s, 1H), 10.65 (s, 1H).

N-[3-(2,2′-Bithiophen-5-yl)-5-(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)acetamide (4m, TMD-375)

To a solution ofN-[3-(2,2′-bithiophen-5-yl)-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11m) (168 mg, crude) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.20 mL, 1.20 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4m (TMD-375)(102 mg, 210 μmol, 49.5% (2 steps)) as a yellow solid. R_(f)=0.44(dichloromethane/methanol=9/1); HPLC retention time 15.3 min; ¹H NMR(400 MHz, DMSO-d₆) 3.57 (s, 2H), 6.66-6.75 (AA′BB′, 2H), 6.85-6.95(AA′BB′, 2H), 7.06-7.17 (m, 4H, includes AA′BB′), 7.34-7.40 (m, 2H),7.53-7.58 (m, 1H), 7.98-8.05 (AA′B′, 2H), 8.85 (s, 1H), 9.30 (s, 1H),9.95 (s, 1H), 10.53 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ 42.0, 115.2(2C), 115.9 (2C), 124.6, 124.9, 125.1, 125.8, 126.3, 128.3 (2C), 128.6,129.0, 130.4 (2C), 136.1, 137.1, 139.4, 139.5, 140.1, 142.1, 148.5,156.3, 159.5, 170.7; IR (KBr, cm⁻¹) 532, 602, 692, 837, 966, 1105, 1165,1223, 1265, 1369, 1450, 1491, 1516, 1609, 1676, 3246, 3385.

2-14) TMD-376 (4n)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-[4-(dimethylamino)phenyl]pyrazin-2-amine(7n)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (500mg, 1.31 mmol) in toluene (15 mL) and ethanol (600 μL) were successivelyadded 4-(dimethylamino)phenylboronic acid (6n) (260 mg, 1.58 mmol),dichlorobis(triphenylphosphine)palladium (II) (56.1 mg, 79.9 μmol) and 1M Na₂CO₃ aqueous solution (1.40 mL, 1.40 mmol) at room temperature,followed by heating to reflux for 24 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (100mL×3). The combined organic extract was washed successively with water(200 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 50 g,n-hexane/ethyl acetate=2/1) to give Compound 7n (488 mg, 1.16 mmol,88.3%) as a yellow solid. R_(f)=0.19 (n-hexane/ethyl acetate=3/1); ¹HNMR (400 MHz, DMSO-d₆) δ 0.17 (s, 6H), 0.92 (s, 9H), 2.94 (s, 6H), 6.02(s, 2H), 6.75-6.83 (AA′BB′, 2H), 6.83-6.92 (AA′BB′, 2H), 7.60-7.70(AA′BB′, 2H), 7.77-7.88 (AA′BB′, 2H), 8.32 (s, 1H); ¹³C NMR (67.8 MHz,DMSO-d₆) δ −4.5 (2C), 17.9, 25.5 (3C), 39.9 (2C), 111.9 (2C), 120.0(2C), 125.0, 126.3 (2C), 128.9 (2C), 130.7, 135.5, 138.5, 139.7, 150.4,151.3, 154.8; IR (KBr, cm⁻¹) 432, 511, 633, 698, 808, 824, 912, 1009,1063, 1103, 1167, 1254, 1360, 1422, 1454, 1510, 1607, 2803, 2857, 2886,2928, 2953, 3179, 3300, 3476.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-(4-(tert-butyldimethylsilyloxy)phenyl)-3-{(4-(dimethylamino)phenyl}pyrazin-2-yl]acetamide(11n)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.00 g, 3.76mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (640 μL, 7.56mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-[4-(dimethylamino)phenyl]pyrazin-2-amine(7n) (315 mg, 749 mol) and 4-(dimethylamino)pyridine (14.8 mg, 121 μmol)dissolved in anhydrous pyridine (15 mL) was added 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) prepared above at 0° C. andthe mixture was heated with stirring at 50° C. for 20 hours. Aftercooling to room temperature, to this was added water and the product wasextracted with ethyl acetate (100 mL×3). The combined organic extractwas washed successively with water (200 mL) and brine (200 mL), followedby drying over anhydrous sodium sulfate. After filtration andconcentration under reduced pressure, the residue was purified by columnchromatography (silica gel 50 g, n-hexane/ethyl acetate=2/1) to giveCompound 11 n (200 mg, 300 μmol, 40.0%) as a yellow solid. R_(f)=0.26(n-hexane/ethyl acetate=2/1); ¹H NMR (400 MHz, DMSO-d₆) 0.17 (s, 6H),0.23 (s, 6H), 0.95 (s, 9H), 0.97 (s, 9H), 2.95 (s, 6H), 3.50 (s, 2H),6.52-6.65 (AA′BB′, 2H), 6.75-6.85 (AA′BB′, 2H), 6.95-7.05 (AA′BB′, 2H),7.10-7.20 (AA′BB′, 2H), 7.56-7.66 (AA′BB′, 2H), 8.04-8.10 (AA′BB′, 2H),8.82 (s, 1H), 10.41 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ −4.5 (2C),−4.6 (2C), 17.9, 18.0, 25.5 (6C), 39.8 (2C), 41.8, 111.3 (2C), 119.5(2C), 120.3 (2C), 124.4, 128.05, 128.07 (2C), 128.9 (2C), 129.2, 130.4(2C), 135.8, 142.0, 147.78, 147.84, 150.5, 153.8, 156.6, 169.3; IR (KBr,cm⁻¹) 527, 685, 781, 839, 914, 1080, 1167, 1256, 1371, 1441, 1508, 1607,1668, 2857, 2928, 2955, 3233

N-[3-{4-(Dimethylamino)phenyl}-5-(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)acetamide(4n, TMD-376)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-{4-(dimethylamino)phenyl}pyrazin-2-yl]acetamide(11n) (148 mg, 220 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.10 mL, 1.10 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4n (TMD-376)(85.3 mg, 194 μmol, 87.8%) as a yellow solid. R_(f)=0.37(dichloromethane/methanol=9/1); HPLC retention time 4.5 min; ¹H NMR (400MHz, DMSO-d₆) δ 2.91 (s, 6H), 3.41 (s, 2H), 6.51-6.59 (AA′BB′, 2H),6.62-6.70 (AA′BB′, 2H), 6.82-6.90 (AA′BB′, 2H), 6.98-7.08 (AA′BB′, 2H),7.53-7.62 (AA′BB′, 2H), 7.94-8.01 (AA′BB′, 2H), 8.74 (s, 1H), 9.25 (s,1H), 9.83 (s, 1H), 10.28 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ 39.8(2C), 41.9, 111.4 (2C), 115.0 (2C), 115.8 (2C), 124.4, 125.3, 126.7,128.1 (2C), 128.9 (2C), 130.3 (2C), 135.5, 141.6, 147.8, 148.3, 150.6,156.2, 159.1, 169.8; IR (KBr, cm⁻¹) 538, 691, 816, 949, 1101, 1169,1250, 1321, 1375, 1437, 1514, 1533, 1609, 1647, 2810, 3005, 3167, 3348.

2-15) TMD-377 (4o)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(4-nitrophenyl)pyrazin-2-amine(7o)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (500mg, 1.31 mmol) in toluene (15 mL) and ethanol (600 μL) were successivelyadded p-nitrophenylboronic acid (6o) (263 mg, 1.58 mmol),dichlorobis(triphenylphosphine)palladium (II) (56.1 mg, 79.9 μmol) and 1M Na₂CO₃ aqueous solution (1.40 mL, 1.40 mmol) at room temperature,followed by heating to reflux for 22 hours. After cooling to roomtemperature, to the mixture was added water and the metal catalyst wasremoved by filtration. The product was extracted with ethyl acetate (100mL×3). The combined organic extract was washed successively with water(200 mL) and brine (300 mL), followed by drying over anhydrous sodiumsulfate. After filtration and concentration under reduced pressure, theresidue was purified by column chromatography (silica gel 50 g,n-hexane/ethyl acetate=3/1) to give Compound 7o (451 mg, 1.07 mmol,81.2%) as a yellow solid. R_(f)=0.19 (n-hexane/ethyl acetate=3/1); ¹HNMR (400 MHz, DMSO-d₆) δ 0.17 (s, 6H), 0.92 (s, 9H), 6.48 (s, 2H),6.85-6.92 (AA′BB′, 2H), 7.82-7.92 (AA′BB′, 2H), 8.02-8.10 (AA′BB′, 2H),8.26-8.36 (AA′BB′, 2H), 8.55 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ −4.5(2C), 18.0, 25.6 (3C), 120.1 (2C), 123.8 (2C), 126.5 (2C), 129.5 (2C),130.0, 134.8, 138.8, 140.1, 144.3, 146.9, 151.8, 155.2; IR (KBr, cm⁻¹)476, 511, 635, 700, 727, 806, 839, 913, 1026, 1084, 1169, 1213, 1279,1348, 1402, 1464, 1516, 1603, 1643, 2859, 2951, 3142, 3300, 3372.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(4-nitrophenyl)pyrazin-2-yl]acetamide(11o)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.00 g, 3.76mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (640 μL, 7.56mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(4-nitrophenyl)pyrazin-2-amine(7o) (317 mg, 751 μmol) and 4-(dimethylamino)pyridine (15.3 mg, 125μmol) dissolved in anhydrous pyridine (15 mL) was added2-[4-(tert-butyldimethylsilyl oxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 20hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=2/1) to give Compound 11o (242 mg, 361 μmol, 48.1%) as a yellowsolid. R_(f)=0.30 (n-hexane/ethyl acetate=2/1); ¹H NMR (400 MHz,DMSO-d₆) δ 0.15 (s, 6H), 0.20 (s, 6H), 0.92 (s, 9H), 0.93 (s, 9H), 3.44(s, 2H), 6.67-6.74 (AA′BB′, 2H), 6.94-7.07 (2AA′BB′, 4H), 7.80-7.86(AA′BB′, 2H), 8.01-8.12 (2AA′BB′, 4H), 9.05 (s, 1H), 10.85 (s, 1H); ¹³CNMR (67.8 MHz, DMSO-d₆) δ −4.5 (2C), −4.6 (2C), 17.9, 18.0, 25.50 (3C),25.51 (3C), 41.6, 119.4 (2C), 120.4 (2C), 123.2 (2C), 127.4, 128.2,128.52 (2C), 128.54 (2C), 130.4 (2C), 138.7, 143.0, 144.5, 145.0, 146.9,147.9, 153.9, 156.9, 168.9; IR (KBr, cm⁻¹) 700, 741, 781, 839, 914,1169, 1265, 1348, 1443, 1508, 1603, 1670, 2857, 2930, 2955.

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(4-nitrophenyl)pyrazin-2-yl]acetamide(4o, TMD-377)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(4-nitrophenyl)pyrazin-2-yl]acetamide(11o) (179 mg, 267 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.40 mL, 1.40 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4o (TMD-377)(98.0 mg, 222 μmol, 83.0%) as an orange solid. R_(f)=0.48(dichloromethane/methanol=9/1); HPLC retention time 7.4 min; ¹H NMR (400MHz, DMSO-d₆) δ 3.40 (s, 2H), 6.60-6.69 (AA′BB′, 2H), 6.86-6.93 (AA′BB′,2H), 6.93-7.03 (AA′BB′, 2H), 7.81-7.89 (AA′BB′, 2H), 8.00-8.10 (2AA′BB′,4H), 9.02 (s, 1H), 9.30 (s, 1H), 9.93 (s, 1H), 10.77 (s, 1H); ¹³C NMR(67.8 MHz, DMSO-d₆) δ 41.7, 115.0 (2C), 115.9 (2C), 123.2 (2C), 124.7,126.1, 128.2 (2C), 128.6 (2C), 130.2 (2C), 138.4, 142.7, 144.5, 145.0,146.9, 148.4, 156.3, 159.3, 169.2; IR (KBr, cm⁻¹) 534, 640, 696, 756,799, 843, 1013, 1080, 1107, 1171, 1236, 1346, 1437, 1516, 1609, 1670,3250.

2-16) TMD-378 (4p)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-(phenylethynyl)pyrazin-2-amine(7p)

Under an argon atmosphere, to a solution of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5)(1.50 g, 3.94 mmol) in diethylamine (20 mL) were successively addedcopper (I) iodide (450 mg, 2.36 mmol), phenylacetylene (6p) (900 μL,8.19 mmol) and dichlorobis(triphenylphosphine)palladium (II) (139 mg,198 μmol) at room temperature and the mixture was stirred for 4 hours.To this was added water and the metal catalyst was removed byfiltration. The product was extracted with ethyl acetate (200 mL×3). Thecombined organic extract was washed successively with water (300 mL) andbrine (300 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 100 g, n-hexane/ethylacetate=3/1) to give Compound 7p (1.56 g, 3.89 mmol, 98.6%) as a brownsolid. R_(f)=0.52 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz,DMSO-d₆) 0.17 (s, 6H), 0.92 (s, 9H), 6.75 (s, 2H), 6.85-6.92 (AA′BB′,2H), 7.38-7.46 (m, 3H), 7.69-7.76 (m, 2H), 7.76-7.83 (AA′BB′, 2H), 8.50(s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ −4.5 (2C), 17.9, 25.5 (3C), 85.4,94.2, 120.1 (2C), 121.6, 121.8, 126.5 (2C), 128.6 (2C), 129.3, 129.9,131.9 (2C), 138.8, 140.0, 154.8, 155.2; IR (KBr, cm⁻¹) 529, 638, 689,754, 781, 806, 824, 912, 1009, 1049, 1070, 1144, 1169, 1258, 1323, 1362,1420, 1454, 1510, 1562, 1605, 2212, 2857, 2928, 2955, 3057, 3173, 3296,3474.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(phenylethynyl)pyrazin-2-yl]acetamide(11p)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.00 g, 3.76mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (640 μL, 7.56mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(phenylethynyl)pyrazin-2-amine (7p) (300 mg, 747 μmol) and 4-(dimethylamino)pyridine (13.8 mg, 113μmol) dissolved in anhydrous pyridine (15 mL) was added2-[4-(tert-butyldimethylsilyl oxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 20hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1) to give Compound 11p (291 mg, 448 μmol, 60.0%) as a brownsolid. R_(f)=0.19 (n-hexane/ethyl acetate=3/1); ¹H NMR (400 MHz,DMSO-d₆) δ 0.10 (s, 6H), 0.20 (s, 6H), 0.90 (s, 9H), 0.93 (s, 9H), 3.65(s, 2H), 6.63-6.73 (AA′BB′, 2H), 6.92-7.02 (AA′BB′, 2H), 7.17-7.27(AA′BB′, 2H), 7.33-7.48 (m, 5H), 7.96-8.05 (AA′BB′, 2H), 8.99 (s, 1H),10.69 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ −4.60 (2C), −4.61 (2C),17.8, 17.9, 25.5 (6C), 41.7, 85.8, 94.5, 119.5 (2C), 120.4 (2C), 121.2,128.2, 128.28 (2C), 128.34, 128.6 (2C), 129.5, 130.3 (2C), 131.8 (2C),133.1, 138.5, 146.6, 148.0, 153.8, 156.9, 169.4; IR (KBr, cm⁻¹) 530,689, 756, 781, 839, 914, 1007, 1125, 1169, 1258, 1379, 1433, 1510, 1605,1672, 2214, 2857, 2930, 2955, 3235.

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-(phenylethynyl)pyrazin-2-yl]acetamide(4p, TMD-378)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-(phenylethynyl)pyrazin-2-yl]acetamide(11p) (198 mg, 305 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.60 mL, 1.60 mmol) at 0° C., and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4p (TMD-378)(93.8 mg, 223 μmol, 73.1%) as a yellow solid. R_(f)=0.48(dichloromethane/methanol=9/1); HPLC retention time 10.0 min; ¹H NMR(400 MHz, DMSO-d₆) δ 3.58 (s, 2H), 6.60-6.70 (AA′BB′, 2H), 6.83-6.93(AA′BB′, 2H), 7.10-7.19 (AA′BB′, 2H), 7.24-7.33 (m, 3H), 7.33-7.49 (m,2H), 7.91-7.98 (AA′BB′, 2H), 8.94 (s, 1H), 9.27 (s, 11H), 9.92 (s, 1H),10.60 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ 41.8, 86.0, 94.4, 115.2(2C), 115.9 (2C), 121.2, 125.6, 125.9, 128.3 (2C), 128.7 (2C), 129.7,130.3 (2C), 131.9 (2C), 133.2, 138.3, 146.3, 148.6, 156.2, 1159.4,169.7; IR (KBr, cm⁻¹) 530, 615, 687, 758, 793, 839, 962, 1125, 1157,1177, 1219, 1267, 1327, 1389, 1456, 1489, 1582, 1684, 2212, 3231, 3391.

2-17) Compound 4q

(Z)-5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-styrylpyrazin-2-amine(7q)

Under an argon atmosphere, to a solution of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(phenylethynyl)pyrazin-2-amine(7p) (100 mg, 249 μmol) in ethyl acetate (5 mL) was added Lindlar'scatalyst (10%6 Pd) (40.0 mg) at room temperature. The atmosphere in thereaction flask was replaced with hydrogen gas and the mixture wasstirred at room temperature for 30 hours. After replacing the atmosphereof the reaction system again with argon, to the mixture was addeddichloromethane (15 mL) and stirred at room temperature for an hour.After removing the catalyst by filtration, the filtrate was washedsuccessively with water (100 mL) and brine (200 mL), followed by dryingover anhydrous sodium sulfate. After filtration and concentration underreduced pressure, the residue was purified by column chromatography(silica gel 10 g, n-hexane/ethyl acetate=3/1) to give the mixture ofCompounds 7q, 7p and 7r (98.2 mg) as a brown oil.

(Z)-2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-{5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-styrylpyrazin-2-yl}acetamide(11q)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (170 rag, 638μmol) in CH₂Cl₂ (5 mL) was added DMF (1 drop) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (110 μL, 1.30mmol) at 0° C. and the mixture was stirred for 30 minutes whileelevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to the mixture (50.3 mg) of 7q, 7p and 7r and4-(dimethylamino)pyridine (3.0 mg, 25 μmol) dissolved in anhydrouspyridine (5 mL) was added 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetylchloride (10) prepared above at 0° C. and the mixture was heated withstirring at 50° C. for 20 hours. After cooling to room temperature, tothis was added water and the product was extracted with ethyl acetate(30 mL×3) The combined organic extract was washed successively withwater (50 mL) and brine (100 mL), followed by drying over anhydroussodium sulfate. After filtration and concentration under reducedpressure, the residue was purified by column chromatography (silica gel10 g, n-hexane/ethyl acetate=3/1) to give the mixture (60.0 mg) ofCompounds 11q, 11p and 11r as a yellow oily substance. R_(f)=0.20(n-hexane/ethyl acetate=3/1).

Compound 4q can be prepared by deprotecting the TBDMS group fromCompound 11q described above according to modifications of the processdescribed in SYNTHESIS EXAMPLES of the other CTMD analogues.

2-18) TMD-379 (4r)

5-[4-(tert-Butyldimethylsilyloxy)phenyl]-3-phenethylpyrazin-2-amine (7r)

Under an argon atmosphere, to a solution of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-(phenylethynyl)pyrazin-2-amine(7p) (400 mg, 996 μmol) in ethyl acetate (20 mL) was addedPalladium/charcoal activated (10% Pd) (80.0 mg) at room temperature. Theatmosphere in the reaction flask was replaced with hydrogen gas and themixture was stirred at room temperature for 3 hours. After replacing theatmosphere of the reaction system again with argon, to the mixture wasadded dichloromethane (15 mL) and stirred at room temperature for anhour. The catalyst was removed by filtration. After concentration underreduced pressure, the residue was purified by column chromatography(silica gel 40 g, n-hexane/ethyl acetate=2/1) to give Compound 7r (403mg, 994 μmol, 99.8%) as a brown oily substance. R_(f)=0.48(n-hexane/ethyl acetate=2/1); ¹H NMR (400 MHz, DMSO-d₆) δ 0.18 (s, 6H),0.93 (s, 9H), 2.92 (t, 3H, J=8.0 Hz), 3.05 (t, 3H, J=8.0 Hz), 6.25 (s,2H), 6.81-6.90 (AA′BB′, 2H), 7.10-7.18 (m, 1H), 7.18-7.35 (m, 4H),7.75-7.85 (AA′BB′, 2H), 8.27 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ −4.5(2C), 18.0, 25.6 (3C), 31.7, 34.0, 120.0 (2C), 125.7, 126.1 (2C), 128.2(2C), 128.5 (2C), 130.9, 135.6, 138.5, 140.1, 141.9, 152.3, 154.6; IR(KBr, cm⁻¹) 507, 635, 677, 698, 750, 781, 841, 914, 1009, 1103, 1146,1167, 1213, 1254, 1389, 1420, 1454, 1512, 1605, 2857, 2928, 2955, 3061,3318.

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-phenethylpyrazin-2-yl]acetamide(11r)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.40 g, 5.26mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (890 μL, 10.5mmol) at 0° C. and the mixture was heated with stirring for 30 minuteswhile elevating to room temperature. The mixture was concentrated underreduced pressure to give crude2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) as acolorless oil, which was used in the following reaction without furtherpurification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]-3-phenethylpyrazin-2-amine (7r)(403 mg, 994 μmol) and 4-(dimethylamino)pyridine (19.6 mg, 160 μmol)dissolved in anhydrous pyridine (15 mL) was added2-[4-(tert-butyldimethylsilyloxy) phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 20hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=3/1) to give Compound 11r (298 mg, 455 μmol, 45.8%) as a yellowoily substance. R_(f)=0.44 (n-hexane/ethyl acetate=2/1); ¹H NMR (400MHz, DMSO-d₆) δ 0.12 (s, 6H), 0.20 (s, 6H), 0.91 (s, 9H), 0.93 (s, 9H),2.83 (t, 1H, J=: 8.4 Hz), 2.94 (t, 1H, J=8.4 Hz), 3.59 (s, 2H),6.72-6.79 (AA′BB′, 2H), 6.92-7.00 (AA′BB′, 2H), 7.01-7.07 (AA′BB′, 2H),7.07-7.15 (m, 1H), 7.16-7.24 (m, 4H), 7.97-8.05 (AA′BB′, 2H), 8.81 (s,1H), 10.44 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ −4.5 (2C), −4.6 (2C),17.9, 18.0, 25.5 (6C), 32.8, 34.7, 41.7, 119.6 (2C), 120.3 (2C), 125.7,128.1 (2C), 128.2 (4C), 128.5, 129.1, 130.2 (2C), 130.3, 136.6, 141.4,143.8, 147.9, 150.6, 153.8, 156.6, 170.3; IR (KBr, cm⁻¹) 525, 698, 781,839, 914, 1007, 1169, 1258, 1362, 1416, 1443, 1510, 1605, 1661, 2857,2930, 2955, 3225.

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)-3-phenethylpyrazin-2-yl]acetamide(4r, TMD-379)

To a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}-3-phenethylpyrazin-2-yl]acetamide(11r) (197 mg, 301 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (1.50 mL, 1.50 mmol) at 0° C. and themixture was stirred for 30 minutes while elevating to room temperature.To the mixture was added saturated NH₄Cl aqueous solution (50 mL) andthe product was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethyl acetate) to give Compound 4r (TMD-379)(100 mg, 236 μmol, 78.3%) as a yellow solid. R_(f)=0.44(dichloromethane/methanol=9/1); HPLC retention time 9.5 min; 1H NMR (400MHz, DMSO-d₆) δ 2.81 (t, 1H, J=8.0 Hz), 2.92 (t, 1H, J=8.0 Hz), 3.52 (s,2H), 6.62-6.72 (AA′BB′, 2H), 6.80-6.90 (AA′BB′, 2H), 6.98-7.05 (AA′BB′,2H), 7.07-7.16 (m, 3H), 7.16-7.24 (m, 2H), 7.90-7.98 (AA′BB′, 2H), 8.76(s, 1H), 9.27 (s, 1H), 9.83 (s, 1H), 10.35 (s, 1H); ¹³C NMR (67.8 MHz,DMSO-d₆) δ 33.0, 34.8, 41.7, 115.2 (2C), 115.8 (2C), 125.8, 126.6, 128.1(2C), 128.26 (2C), 128.29 (2C), 128.6, 130.1 (2C), 136.4, 141.5, 143.4,148.5, 150.7, 156.2, 159.0, 170.6; IR (KBr, cm⁻¹) 519, 606, 692, 839,966, 1157, 1223, 1265, 1373, 1452, 1516, 1595, 1674, 3273.

2-19) TMD-348 (4s)

2-[4-(tert-Butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]acetamide(11s)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.00 mL, 11.8mmol) at 0° C. and the mixture was stirred for an hour while elevatingto room temperature. The mixture was concentrated under reduced pressureto give crude 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride(10) as a colorless oil, which was used in the following reactionwithout further purification.

Under an argon atmosphere, to a mixture of5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (7s) (350 mg,1.16 mmol) and 4-(dimethylamino)pyridine (15.0 mg, 123 μmol) dissolvedin anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 20hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=7/1) to give Compound 1 is (478 mg, 869 μmol, 74.8%) as a yellowfoamy solid. R_(f)=0.19 (n-hexane/ethyl acetate=6/1); ¹H NMR (400 MHz,DMSO-d₆) δ 0.14 (s, 6H), 0.19 (s, 6H), 0.91 (s, 9H), 0.94 (s, 9H), 3.66(s, 2H), 6.75-6.82 (AA′BB′, 2H), 6.91-6.97 (AA′BB′, 2H), 7.17-7.24(AA′BB′, 2H), 7.93-8.00 (AA′BB′, 2H), 8.90 (d, 1H, J=1.4 Hz), 9.28 (d,1H, J=1.4 Hz), 10.99 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ −4.6 (4C),17.8, 17.9, 25.46 (3C), 25.47 (3C), 41.8, 119.6 (2C), 120.2 (2C), 127.5(2C), 128.2, 129.2, 130.4 (2C), 134.9, 138.7, 146.3, 147.0, 153.8,156.2, 170.3; IR (KBr, cm⁻¹) 417, 517, 575, 638, 681, 708, 781, 806,839, 914, 1009, 1051, 1103, 1167, 1260, 1354, 1416, 1468, 1508, 1545,1605, 1670, 2857, 2886, 2930, 2955, 3034, 3063; HRMS (ESI⁺) m/z 550.2930([M+H], C₃₀H₄₄N₃O₃Si₂ ⁺ requires 550.2916).

2-(4-Hydroxyphenyl)-N-[5-(4-hydroxyphenyl)pyrazin-2-yl]acetamide (4s,TMD-348)

To a solution of2-[4-(ter-butyldimethylsilyloxy)phenyl]-N-[5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]acetamide(11s) (300 mg, 546 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (2.80 mL, 2.80 mmol) at 0° C. and themixture was stirred for an hour while elevating to room temperature. Tothe mixture was added saturated NH₄Cl aqueous solution (50 mL) and theproduct was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethanol) to give Compound 4s (TMD-348) (191 mg,422 μmol, 95.8%) as a brown solid. R=0.51(dichloromethane/methanol=9/1); HPLC retention time 4.9 min; ¹H NMR (400MHz, DMSO-d₆) 3.57 (s, 1H), 6.62-6.70 (AA′BB′, 2H), 6.79-6.84 (AA′BB′,2H), 7.06-7.12 (AA′BB′, 2H), 7.83-7.92 (AA′BB′, 2H), 8.82 (d, 1H, J=1.4Hz), 9.22 (d, 1H, J=1.4 Hz), 9.23 (s, 1H), 9.75 (s, 1H), 10.86 (s, 1H);¹³C NMR (75.5 MHz, DMSO-d₆) δ 41.9, 115.2 (2C), δ 115.8 (2C), 125.7,126.8, 127.7 (2C), 130.3 (2C), 134.9, 138.4, 146.7, 146.9, 156.3, 158.7,170.7; IR (KBr, cm⁻¹) 419, 519, 613, 681, 804, 837, 910, 1020, 1051,1107, 1171, 1252, 1356, 1443, 1508, 1541, 1611, 1676, 3026, 3354; HRMS(FAB⁺/glycerol) m/z 322.1187 ([M+H]⁺, C₁₈H₁₆N₃O₃ ⁺ requires 322.1192).

2-20) TMD-349 (4t)

N-[3-Bromo-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11t)

Under an argon atmosphere, to a solution of2-[4-(tert-butyldimethylsilyloxy)phenyl]acetic acid (9) (1.55 g, 5.82mmol) in CH₂Cl₂ (10 mL) was added DMF (3 drops) by a Pasteur pipette atroom temperature. To this was added oxalyl dichloride (1.00 mL, 11.8mmol) at 0° C. and the mixture was stirred for an hour while elevatingto room temperature, The mixture was concentrated under reduced pressureto give crude 2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride(10) as a colorless oil, which was used in the following reactionwithout further purification.

Under an argon atmosphere, to the mixture of3-bromo-5-[4-(tert-butyldimethylsilyloxy)phenyl]pyrazin-2-amine (5) (443mg, 1.16 mmol) and 4-(dimethylamino)pyridine (15.2 mg, 124 mol)dissolved in anhydrous pyridine (20 mL) was added2-[4-(tert-butyldimethylsilyloxy)phenyl]acetyl chloride (10) preparedabove at 0° C. and the mixture was heated with stirring at 50° C. for 19hours. After cooling to room temperature, to this was added water andthe product was extracted with ethyl acetate (200 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue waspurified by column chromatography (silica gel 50 g, n-hexane/ethylacetate=4/1) to give Compound 11t (431 mg, 685 mol, 58.9%) as a yellowfoamy solid. R_(f)=0.22 (n-hexane/diethyl ether=1/1); ¹H NMR (400 MHz,DMSO-d₆) δ 0.15 (s, 6H), 0.21 (s, 6H), 0.91 (s, 9H), 0.94 (s, 9H), 3.61(s, 2H), 6.74-6.82 (AA′BB′, 2H), 6.94-7.01 (AA′BB′, 2H), 7.16-7.23 (AABB′, 2H), 7.94-8.01 (AA′BB′, 2H), 9.03 (s, 1l), 10.54 (s, 1H); 13C NMR(75.5 MHz, DMSO-d₆) δ −4.6 (4C), 17.89, 17.93, 25.46 (3C), 25.52 (3C),41.5, 119.6 (2C), 120.5 (2C), 127.3, 128.1, 128.4 (2C), 130.4 (2C),136.3, 138.0, 144.6, 149.1, 153.8, 157.2, 169.8; IR (KBr, cm⁻¹) 467,521, 623, 685, 716, 781, 802, 839, 914, 1007, 1047, 1078, 1107, 1169,1256, 1333, 1362, 1412, 1441, 1474, 1508, 1566, 1605, 1672, 2857, 2886,2930, 2955, 3032, 3057, 3231; HRMS (ESI⁺) m/z 650.1839 ([M+Na]⁺,C₃₀H₄₂BrN₃NaO₃Si₂ requires 650.1840).

N-[3-Bromo-5-(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)acetamide(4t, TMD-349)

To a solution ofN-[3-bromo-5-{4-(tert-butyldimethylsilyloxy)phenyl}pyrazin-2-yl]-2-[4-(tert-butyldimethylsilyloxy)phenyl]acetamide(11t) (290 mg, 461 μmol) in THF (5 mL) was added tetrabutylammoniumfluoride (1.0 M THF solution) (2.30 mL, 2.30 mmol) at 0° C., and themixture was stirred for an hour while elevating to room temperature. Tothe mixture was added saturated NH₄Cl aqueous solution (50 mL) and theproduct was extracted with ethyl acetate (100 mL×3). The combinedorganic extract was washed successively with water (200 mL) and brine(200 mL), followed by drying over anhydrous sodium sulfate. Afterfiltration and concentration under reduced pressure, the residue wasrecrystallized (n-hexane/ethanol) to give Compound 4t (TMD-349) (147 mg,367 μmol, 79.5%) as a brown solid. R_(f)=0.50(dichloromethane/methanol=9/1); HPLC retention time 5.5 min; ¹H NMR (400MHz, DMSO-d₆) δ 3.54 (s, 2H), 6.65-6.71 (AA′BB′, 2H), 6.83-6.89 (AA′BB′,2H), 7.08-7.14 (AA′BB′, 2H), 7.87-7.95 (AA′BB′, 2H), 8.97 (s, 1H), 9.25(s, 1H), 10.00 (s, 1H), 10.45 (s, 1H); ¹³C NMR (75.5 MHz, DMSO-d₆) δ41.5, 115.2 (2C), 116.0 (2C), 124.8, 125.4, 128.5 (2C), 130.3 (2C),136.6, 137.7, 144.2, 149.8, 156.2, 159.8, 170.2; IR (KBr, cm⁻¹) 519,623, 795, 839, 1047, 1080, 1173, 1229, 1271, 1339, 1449, 1483, 1516,1609, 1676, 3275; HRMS (ESI⁺) m/z 422.0119 ([M+Na]⁺, C₁₈H₁₄BrN₃NaO₃ ⁺requires 422.0111).

Synthesis Example 3 Coelenteramide (CTMD) Analogues Modified at the C-2Position 3-1) TMD-331 (4u)

1-[3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-3-(4-methoxyphenyl)urea(11u)

Under an argon atmosphere, to a solution of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (12) (synthesized by theprocess of M. Adamczyk, et al., Org. Prep. Proced Int., 33, 477-485(2001)) (306 mg, 1.05 mmol) in anhydrous 1,2-dichloroethane (10 mL) wasadded 4-methoxyphenyl isocyanate (13) (191 μL, 1.00 mmol) at roomtemperature, and the mixture was heated with stirring at 80° C. for 19hours. After cooling to room temperature, the solid precipitated wascollected by filtration and dried in vacuo to give the crude product(293 mg, <664 μmol, <63.2%) as a colorless solid. An aliquot (93.0 mg,<211 μmol) was recrystallized from methanol to give Compound 11u (54.9mg, 125 μmol, 59.2%) as a colorless solid. R_(f)=0.50 (n-hexane/ethylacetate=1/1); ¹H NMR (400 MHz, DMSO-dc) δ 3.73 (s, 3H), 3.81 (s, 3H),4.36 (s, 2H), 6.98-6.93 (AA′BB′, 2H), 7.02-7.08 (AA′BB′, 2H), 7.17-7.25(m, 1H), 7.28-7.32 (m, 4H), 7.43-7.48 (AA′BB′, 2H), 7.95-8.01 (AA′BB′,2H), 8.79 (s, 1H), 9.04 (s, 1H), 10.58 (s, 1H); ¹³C NMR (67.8 MHz,DMSO-d₆) 38.5, 55.18, 55.24, 114.0 (2C), 114.4 (2C), 121.1 (2C), 126.3,127.2 (2C), 128.2, 128.3 (2C), 129.0 (2C), 131.7, 134.5, 137.9, 144.4,145.0, 145.4, 152.2, 155.1, 160.1; IR (KBr, cm⁻¹) 704, 746, 824, 1038,1173, 1242, 1329, 1369, 1416, 1481, 1510, 1570, 1607, 1668, 2833, 3030,3261; Anal. Calcd. For C₂₆H₂₄N₄O₃: C, 70.89; H, 5.49; N, 12.72. Found:C, 70.66; H, 5.49; N, 12.61.

1-[3-Benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl]-3-(4-hydroxyphenyl)urea(4u, TMD-331)

Under an argon atmosphere, to a solution of1-[3-benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-3-(4-methoxy phenyl)urea(11u) (200 mg, 454 μmol) in anhydrous dichloromethane (10 mL) was addedboron tribromide (1.0 M dichloromethane solution, 2.27 mL, 2.27 mmol) atroom temperature, and the mixture was heated to reflux for 24 hours.After cooling to room temperature, to this was added saturated aqueoussodium bicarbonate solution and the mixture was concentrated underreduced pressure using a rotary evaporator to remove dichloromethane.The solid was collected by filtration and dried in vacuo to give thecrude product (180 mg) as a colorless solid. The solid wasrecrystallized from ethyl acetate/methanol to give Compound 4u (TMD-331)(98.2 mg, 238 μmol, 52.4%) as a colorless solid. R_(f)=0.66(n-hexane/ethyl acetate=1/2); ¹H NMR (400 MHz, DMSO-d₆) δ 4.35 (s, 2H),6.69-6.74 (AA′BB′, 2H), 6.84-6.88 (AA′BB′, 2H), 7.17-7.24 (m, 1H),7.27-7.33 (m, 5H), 7.84-7.89 (AA′BB′, 2H), 8.72 (s. 1H), 8.95 (s, 1H),9.19 (s, 1H), 9.79 (s, 1H), 10.49 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ38.5, 115.3 (2C), 115.7 (2C), 121.5 (2C), 126.3, 126.7, 127.4 (2C),128.4 (2C), 128.9 (2C), 130.1, 134.2, 138.0, 144.8, 145.2, 152.3, 153.3,158.5; IR (KBr, cm⁻¹) 519, 637, 702, 750, 835, 1105, 1171, 1223, 1246,1369, 1445, 1481, 1508, 1578, 1609, 1681, 3035, 3256; Anal. Calcd. ForC₂₄H₂₀N₄O₃: C, 69.89; H, 4.89; N, 13.58. Found: C, 69.58; H, 5.05; N,13.37.

3-2) TMD-332 (4v)

1-[3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-3-(4-methoxyphenyl)thiourea(11v)

Under an argon atmosphere, to a solution of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (12) (synthesized by theprocess of M. Adamczyk, et al., Org. Prep. Proced Int., 33, 477-485(2001)) (291 mg, 1.00 mmol) in anhydrous DMF (5 mL) was added sodiumhydride (43.6 mg, 1.00 mmol) at room temperature. To the mixture wasadded a solution of 4-methoxyphenyl isothiocyanate (14) (182 mg, 1.10mmol) dissolved in anhydrous DMF (2 mL) at 0° C. and stirred for 5.5hours at room temperature. To this was added aqueous 2 M HCl solutionand the resulting solid was collected by filtration. The crude productwas purified by column chromatography (silica gel 30 g, n-hexane/ethylacetate/dichloromethane=57/3/40) to give the product as a yellow solidcontaining some impurities (327 mg, <716 μmol). The product wasrecrystallized from ethyl acetate to give Compound 11v (255 mg, 559μmol, 55.9%) as a yellow solid. R=0.24 (n-hexane/ethyl acetate=4/1); ¹HNMR (400 MHz, DMSO-d₆) δ 3.75 (s, 3H), 3.82 (s, 3H), 4.42 (s, 2H),6.90-6.96 (AA′BB′, 2H), 7.03-7.09 (AA′BB′, 2H), 7.20-7.26 (m, 1H),7.30-7.36 (m, 4H), 7.38-7.45 (AA′BB′, 2H), 7.99-8.05 (AA′BB′, 2H), 8.83(s, 1H), 9.65 (s, 1H), 11.76 (br s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ38.9, 55.2 (2C), 113.7 (2C), 114.4 (2C), 126.2 (2C), 126.5, 127.5 (2C),128.0, 128.5 (2C), 129.0 (3C), 131.6, 134.7, 137.5, 144.9, 145.7, 157.1,160.4, 179.0; IR (KBr, cm⁻¹) 581, 700, 741, 799, 833, 897, 1022, 1070,1146, 1248, 1283, 1333, 2037, 2351, 2835, 2988, 3395; Anal. Calcd. ForC₂₆H₂₄N₄O₂S: C, 68.40; H, 5.30; N, 12.27. Found: C, 68.47; H, 5.20; N,12.26.

1-[3-Benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl]-3-(4-hydroxyphenyl)thiourea(4v, TMD-332)

Under an argon atmosphere, to a solution of1-[3-benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-3-(4-methoxyphenyl)thiourea (11v) (136 mg, 299 μmol) in anhydrous dichloromethane (7mL) was added boron tribromide (1.0 M dichloromethane solution, 1.50 mL,1.50 mmol) at room temperature, and the mixture was heated to reflux for18 hours. After cooling to room temperature, to this was added saturatedaqueous sodium bicarbonate solution and the mixture was concentratedunder reduced pressure using a rotary evaporator to removedichloromethane. The solid was collected by filtration and dried invacuo to give the crude product (126 mg) as an orange solid. The solidwas recrystallized from ethyl acetate to give Compound 4v (TMD-332)(80.6 mg, 188 μmol, 62.9%) as an orange solid. R_(f)=0.48(n-hexane/ethyl acetate=1/2); ¹H NMR (400 MHz, DMSO-d₆) δ 4.40 (s, 2H),6.70-6.76 (AA′BB′, 2H), 6.84-6.90 (AA′BB′, 2H), 7.20-7.35 (m, 7H),7.88-7.94 (AA′BB′, 2H), 8.76 (s, 1H), 9.47 (s, 1H), 9.53 (s, 1H), 9.85(s, 1H), 11.71 (br s, 1H); IR (KBr, cm⁻¹) 542, 584, 700, 741, 837, 1157,1215, 1263, 1342, 1395, 1449, 1510, 1578, 1609, 2941, 3032, 3273, 3406;HRMS (ESI⁺) m/z 429.1382 ([M+H]⁺, C₂₄H₂₁N₄O₂S requires 429.1380).

3-3) TMD-330 (4w)

N-[3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-2-(4-methoxyphenyl)propanamide(11w)

Under an argon atmosphere, 2-(4-methoxyphenyl)propanoic acid(synthesized by the process of I. Shiina, et al., Eur J. Org. Chem.,5887-5890 (2008)) (1.20 g, 6.66 mmol) was dissolved in thionyl chloride(6.00 mL, 82.6 mmol) and the solution was heated to reflux for 27 hours.After cooling to room temperature, the mixture was concentrated underreduced pressure to give the crude product of2-(4-methoxyphenyl)propanoyl chloride (16) as a colorless oil. Theproduct was used in the following reaction without further purification.

Under an argon atmosphere, to a solution of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (12) (synthesized by theprocess of M. Adamczyk, et al., Org. Prep. Proced Int., 33, 477-485(2001)) (500 mg, 1.72 mmol) in pyridine (8 mL) were successively added4-(dimethylamino)pyridine (21.0 mg, 172 μmol) and2-(4-methoxyphenyl)propanoyl chloride (16) prepared above at roomtemperature, and the mixture was heated with stirring at 55° C. for 14hours. After cooling to room temperature, to the mixture was added waterand the product was extracted with dichloromethane (×3). The combinedorganic extract was dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The residual pyridine wasazeotropically removed with toluene (×3). The residue was purified twiceby column chromatography (silica gel 50 g, dichloromethane/ethylacetate=9/1, and silica gel 50 g, n-hexane/ethyl acetate=9/1→6/1) togive Compound 11w (942 mg, <quant.) as a colorless foamy solidcontaining some impurities.

N-[3-Benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl]-2-(4-hydroxyphenyl)propanamide(4w, TMD-330)

Under an argon atmosphere, to a solution of the crude product ofN-[3-benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-2-(4-methoxyphenyl)propanamide(11w) (500 mg, <1.10 mmol) in anhydrous dichloromethane (20 mL) wasadded boron tribromide (1.0 M dichloromethane solution, 5.50 mL, 5.50mmol) at room temperature, and the mixture was heated to reflux for 21hours. After cooling to room temperature, to this was added saturatedaqueous sodium bicarbonate solution and the mixture was concentratedunder reduced pressure using a rotary evaporator to removedichloromethane. The solid was collected by filtration and dried invacuo to give the crude product (330 mg) as a brown solid. The solid wasrecrystallized from methanol to give Compound 4w (TMD-330) (112 mg, 263μmol, <23.9% (2 steps)) as a colorless solid. R_(f)=0.26 (n-hexane/ethylacetate=1/1); ¹H NMR (400 MHz, DMSO-d₆) δ 1.37 (d, 3H, J=7.0 Hz), 3.81(q, 1H, J=7.0 Hz), 3.84-4.00 (m, 2H), 6.72-6.79 (AA′BB′, 2H), 6.85-6.96(m, 4H), 7.09-7.19 (m, 3H), 7.21-7.27 (AA′BB′, 2H), 7.90-7.98 (AA′BB′,2H), 8.79 (s, 1H), 9.31 (s, 1H), 9.87 (s, 1H), 10.30 (s, 1H); ¹³C NMR(67.8 MHz, DMSO-d₆) δ 18.3, 44.3, 115.2 (2C), 115.7 (2C), 126.1, 126.5,128.0 (2C), 128.1 (2C), 128.4 (2C), 128.9 (2C), 131.6, 136.8, 138.3,143.3, 148.6, 150.6, 156.3, 159.1, 173.4 (one carbon at the benzylposition was unobservable due to overlapping with the septet peak ofDMSO); IR (KBr, cm⁻¹) 530, 596, 700, 725, 837, 924, 1171, 1225, 1267,1319, 1375, 1450, 1487, 1545, 1611, 1670, 2976, 3310; HRMS (ESI⁺) m/z448.1644 ([M+Na]+, C₂₆H₂₃N₃NaO₃ ⁺ requires 448.1632).

3-4) TMD-365 (4x)

N-[3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-4-methoxybenzamide (11x)

To a solution of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (12)(synthesized by the process of M. Adamczyk, et al., Org. Prep. ProcedInt., 33, 477-485 (2001)) (500 mg, 1.72 mmol) in pyridine (5 mL) weresuccessively added 4-(dimethylamino)pyridine (21.0 mg, 172 μmol) and4-methoxybenzoyl chloride (17) (587 mg, 3.44 mmol) at room temperature,and the mixture was heated with stirring at 50° C. for 17 hours. Aftercooling to room temperature, to the mixture was added water and theproduct was extracted with dichloromethane (×3). The combined organicextract was dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The residual pyridine wasazeotropically removed with toluene (×3). The residue was purified bycolumn chromatography (silica gel 50g, dichloromethane/ethylacetate=9/1). The residue was further recrystallized from ethyl acetateto give Compound 11x (592 mg, 1.39 mmol, 80.8%) as a colorless solid.R_(f)=0.63 (dichloromethane/ethyl acetate=9/1); ¹H NMR (400 MHz,DMSO-d₆) δ 3.83 (s, 3H), 3.85 (s, 3H), 4.17 (s, 2H), 7.04-7.11 (m, 4H),7.14-7.26 (m, 5H), 7.93-7.98 (AA′BB′, 2H), 8.05-8.11 (AA′BB′, 2H), 8.96(s, 1H), 10.63 (s, 1H); ¹³C NMR (67.8 MHz, DMSO+CDCl₃) δ 40.3, 54.7,54.8, 112.9 (2C), 113.7 (2C), 125.4, 125.7, 127.4 (2C), 127.7 (2C),128.0, 128.5 (2C), 129.4 (2C), 136.2, 137.6, 143.6, 148.2, 150.4, 160.1,162.0, 165.5; IR (KBr, cm⁻¹) 700, 743, 843, 1030, 1159, 1177, 1258,1290, 1452, 1485, 1514, 1535, 1580, 1609, 1643, 3242; Anal. Calcd. ForC₂₆H₂₃N₃O₃: C, 73.39; H, 5.45; N, 9.88. Found: C, 73.48; H, 5.40; N,9.94.

N-[3-Benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl]-4-hydroxybenzamide (4x,TMD-365)

Under an argon atmosphere, to a solution ofN-[3-benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-4-methoxybenzamide (11x)(300 mg, 705 μmol) in anhydrous dichloromethane (15 mL) was added borontribromide (1.0 M dichloromethane solution, 3.53 mL, 3.53 mmol) at roomtemperature, and the mixture was was heated to reflux for 17 hours.After cooling to room temperature, to this was added saturated aqueoussodium bicarbonate solution and the mixture was concentrated underreduced pressure using a rotary evaporator to remove dichloromethane.The solid was collected by filteration and dried in vacuo to give thecrude product (288 mg) as a brown solid. The solid was purified bycolumn chromatography (silica gel 30 g, n-hexane/ethyl acetate=2/3). Theproduct was further recrystallized from ethyl acetate to give Compound4x (TMD-365) (119 mg, 300 μmol, 42.6%) as a yellow solid. R_(f)=0.46(dichloromethane/methanol=9/1); ¹H NMR (400 MHz, DMSO-d₆) δ 4.14 (s,2H), 6.83-6.92 (m, 4H), 7.12-7.27 (m, 5H), 7.81-7.88 (AA′BB′, 2H),7.93-7.99 (AA′BB′, 2H), 8.88 (s, 1H), 9.89 (br s, 1H), 10.19 (br s, 1H),10.49 (s, 1H); ¹³C NMR (67.8 MHz, DMSO-d₆) δ 40.0, 115.0 (2C), 115.8(2C), 124.1, 126.2, 126.5, 128.1 (2C), 128.2 (2C), 129.0 (2C), 130.1(2C), 136.9, 138.4, 144.1, 148.6, 151.3, 159.1, 161.0, 165.9; IR (KBr,cm⁻¹) 621, 708, 754, 835, 1172, 1215, 1248, 1284, 1368, 1394, 1439,1485, 1601, 1655, 3030, 3298.

3-5) TMD-366 (4y)

N-[3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-3-methoxybenzamide (11y)

To a solution of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (12)(synthesized by the process of M. Adamczyk, et al., Org. Prep. ProcedInt., 33, 477-485 (2001)) (800 mg, 2.75 mmol) in pyridine (10 mL) weresuccessively added 4-(dimethylamino)pyridine (33.6 mg, 275 μmol) andm-anisoyl chloride (18) (773 μL, 5.50 mmol) at room temperature, and themixture was heated with stirring at 50° C. for 22 hours. After coolingto room temperature, to the mixture was added water and the product wasextracted with dichloromethane (×3). The combined organic extract wasdried over anhydrous sodium sulfate. After filtration and concentrationunder reduced pressure, the residual pyridine was azeotropically removedwith toluene (×3). The residue was purified by column chromatography(silica gel 100 g, dichloromethane/ethyl acetate=9/1). The residue wasrecrystallized from ethyl acetate to give Compound 11y (967 mg, 2.27mmol, 82.5%) as a colorless solid. R_(f)=0.63 (dichloromethane/ethylacetate=9/1), ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 6H), 4.19 (s, 2H),7.05-7.12 (AA′BB′, 2H), 7.14-7.28 (m, 6H), 7.42-7.49 (m, 2H), 7.53 (d,1H, J=7.5 Hz), 8.06-8.12 (AA′BB′, 2H), 8.98 (s, 1H), 10.77 (s, 1H); ¹³CNMR (67.8 MHz, CDCl₃) δ 41.5, 55.3, 55.4, 112.5, 114.3 (2C), 118.6,119.3, 126.7 (2C), 128.1, 128.55, 128.61 (2C), 128.5 (2C), 129.6, 135.0,136.8, 137.7, 143.3, 149.2, 149.9, 159.8, 160.9, 165.7; IR (KBr, cm⁻¹)700, 746, 833, 1022, 1040, 1117, 1179, 1254, 1300, 1325, 1373, 1416,1441, 1501, 1607, 1655, 2936, 3265; Anal. Calcd. For C₂₆H₂₃N₃O₃: C,73.39; H, 5.45; N, 9.88. Found: C, 73.55; H, 5.36; N, 9.86.

N-[3-Benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl]-3-hydroxybenzamide (4y,TMD-366)

Under an argon atmosphere, to a solution ofN-(3-benzyl-5-(4-methoxyphenyl)pyrazin-2-yl)-3-methoxybenzamide (11y)(300 mg, 705 μmol) in anhydrous dichloromethane (15 mL) was added borontribromide (1.0 M dichloromethane solution, 3.53 mL, 3.53 mmol) at roomtemperature, and the mixture was heated to reflux for 19 hours. Aftercooling to room temperature, to this was added saturated aqueous sodiumbicarbonate solution and the mixture was concentrated under reducedpressure using a rotary evaporator to remove dichloromethane. The solidwas collected by filteration and dried in vacuo to give the crudeproduct (343 mg) as an orange solid. The solid was purified by columnchromatography (silica gel 50 g, n-hexane/ethyl acetate=2/3). Theproduct was further recrystallized from ethyl acetate to give Compound4y (TMD-366) (193 mg, 486 μmol, 68.9%) as a pale yellow solid.R_(f)=0.32 (n-hexane/ethyl acetate=1/2); ¹H NMR (400 MHz, DMSO-d₆) δ4.16 (s, 2H), 6.86-6.92 (AA′BB′, 2H), 6.98-7.03 (m, 1H), 7.14-7.27 (m,5H), 7.29-7.40 (m, 3H), 7.94-8.00 (AA′BB′, 2H), 8.90 (s, 1H), 9.78 (brs, 1H), 9.90 (br s, 1H), 10.65 (s, 1H), ¹³C NMR (67.8 MHz, DMSO-d₆) δ114.8, 115.8 (2C), 118.4, 119.0, 126.2, 126.4, 128.1 (2C), 128.3 (2C),129.0 (2C), 129.5, 135.0, 137.0, 138.3, 143.8, 148.9, 151.3, 157.4,159.1, 166.3 (one carbon at the benzyl position was unobservable due tooverlapping with the septet peak of DMSO); IR (KBr, cm⁻¹) 596, 683, 708,748, 843, 1165, 1209, 1250, 1269, 1304, 1371, 1443, 1503, 1520, 1595,1649, 3254.

3-6) TMD-368 (4z)

N-[3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-6-methoxy-N-(6-methoxy-1-naphthoyl)-1-naphthamide(11z)

Under an argon atmosphere, 6-methoxy-1-naphthoic acid (synthesized bythe process of J. D. Moseley and J. P. Gilday, Tetrahedron, 62,4690-4697 (2006)) (1.20 g, 5.93 mmol) was dissolved in thionyl chloride(5.00 mL, 68.8 mmol) and the solution was heated to reflux for 3 hours.After cooling to room temperature, the mixture was concentrated underreduced pressure to give the crude product of 6-methoxy-1-naphthoylchloride (22) as a colorless oil. The product was used in the followingreaction without further purification.

Under an argon atmosphere, to a solution of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (12) (synthesized by theprocess of M. Adamczyk, et al., Org. Prep. Proced Int., 33, 477-485(2001)) (500 mg, 1.72 mmol) in pyridine (5 mL) were successively added4-(dimethylamino)pyridine (21.0 mg, 172 μmol) and 6-methoxy-1-naphthoylchloride (22) prepared above at room temperature, and the mixture washeated with stirring at 50° C. 18 hours. After cooling to roomtemperature, to the mixture was added water and the product wasextracted with dichloromethane (×3). The combined organic extract wasdried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure. The residual pyridine was azeotropically removed withtoluene (×3). The residue was successively purified twice by columnchromatography (silica gel 50 g, dichloromethane/ethyl acetate=9/1, andsilica gel 50 g, n-hexane/ethyl acetate=2/1) to give Compound 11z (775mg, 1.17 mmol, <68.3%) as a brown foamy solid containing someimpurities. R_(f)=0.21 (n-hexane/ethyl acetate=2/1); ¹H NMR (400 MHz,DMSO-d₆) δ 3.82 (s, 3H), 3.83 (s, 6H), 4.39 (s, 2H), 7.04-7.09 (AA′BB′,2H), 7.15-7.23 (m, 6H), 7.26-7.31 (m, 1H), 7.34-7.41 (m, 4H), 7.63-7.69(m, 2H), 7.75-7.80 (AA′BB′, 2H), 8.02-8.08 (m, 4H), 9.07 (s, 1H).

N-[3-Benzyl-5-(4-hydroxyphenyl)pyrazin-2-yl]-6-hydroxy-1-naphthamide(4z, TMD-368)

Under an argon atmosphere, to a solution of the crude product ofN-[3-benzyl-5-(4-methoxyphenyl)pyrazin-2-yl]-6-methoxy-N-(6-methoxy-1-naphthoyl)-1-naphthamide(11z) (500 mg, <758 μmol) in anhydrous dichloromethane (15 mL) was addedboron tribromide (1.0 M dichloromethane solution, 3.15 mL, 3.15 mmol) atroom temperature, and the mixture was heated to reflux for 18 hours.After cooling to room temperature, to this was added saturated aqueoussodium bicarbonate solution. The solid in the suspension obtained duringconcentration under reduced pressure was collected by filtration anddried to give the crude product (294 mg, 657 μmol, <86.7%) as a brownsolid. The product was recrystallized twice from ethyl acetate to giveCompound 4z (TMD-368) (10.0 mg, 22.3 μmol, 2.9%) as a pale yellow solid.R_(f)=0.46 (n-hexane/ethyl acetate=1/2); ¹H NMR (400 MHz, DMSO-d₆) δ4.32 (s, 2H), 6.85-6.94 (AA′BB′, 2H), 7.10-7.16 (m, 1H), 7.17-7.33 (m,6H), 7.34-7.40 (m, 1H), 7.41-7.47 (m, 1H), 7.82-7.88 (m, 1H), 7.94-8.02(AA′BB′, 2H), 8.04-8.09 (m, 1H), 8.92 (s, 1H), 9.91 (s, 2H), 10.83 (s,1H).

Hereinafter, Compounds 3a, 3b and 3d to 3j in SYNTHESIS EXAMPLESdescribed above are sometimes simply referred to as “coelenterazineanalogues” and Compounds 4a to 4z as “coelenteramide analogues.”

Example 1 Preparation of Semi-Synthetic Aequorins for SubstrateSpecificity Analysis and Determination of Luminescence Activity

In preparing the following semi-synthetic aequorin, recombinantapoaequorin manufactured by Chisso Corporation was used. Thisrecombinant apoaequorin was expressed and purified according to themethod described in Inouye, S. and Hosoya. T Biochem. Biophys. Res.Commun (2009) 386: 612-622.

(1) Preparation of Semi-Synthetic Aequorins

To 1 ml of 30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA, 1 μl of2-mercaptoethanol and 1.31 μg of the recombinant apoaequorin solution(manufactured by Chisso Corporation) were added and mixed. Subsequently,1 μl of coelenterazine or its analogues dissolved in ethanol was addedto the mixture. The mixture was allowed to stand at 4° C. for 18 hoursto convert into semi-synthetic aequorins.

(2) Assay for Luminescence Activity

Specifically, the luminescence activity described above was assayed asfollows. To 2 μl of a solution of semi-synthetic aequorin in eachregeneration process was added 100 μl of 50 mM Tris-HCl (pH 7.6)containing 50 mM calcium chloride solution, whereby the luminescencereaction was triggered. The luminescence activity was measured for 10seconds on a luminometer luminescencer-PSN AB2200 (manufactured by AttoCo., Ltd.) to obtain the maximum intensity (I_(max)) of luminescenceactivity. Also, the luminescence capacity of semi-synthetic aequorinswas obtained by integrating for 10 seconds.

The results are summarized in TABLE 1 below. Semi-synthetic aequorinsincluding coelenterazine analogues had sufficient luminescenceintensities to detect the luminescence activity, though the maximumluminescence intensity was low.

Example 2 Measurement of Luminescence Patterns of Semi-SyntheticAequorins and Determination of Half Decay Time

A solution of the regenerated semi-synthetic aequorin was diluted to10-fold with 20 mM Tris-HCl (pH 7.6) containing 0.1% BSA (manufacturedby Sigma, Inc.), 0.01 mM EDTA and 150 mM NaCl. The dilution wasdispensed into a 96-well microplate (Nunc #236108) at 5 μl/well. Theluminescence reaction was triggered by injecting 50 mM Tris-HCl (pH 7.6)containing 50 mM calcium chloride solution at 100 μl/well. Theluminescence patterns for 60 seconds were measured to determine halfdecay time of the luminescence (a time period in which the luminescencebecomes half of the maximum luminescence intensity).

The results are summarized in TABLE 1. The results reveal thatsemi-synthetic aequorins prepared from coelenterazine analogues had alonger half-decay time as compared to the half-decay time (0.81 second)of native aequorin prepared from coelenterazine; in particular, thehalf-decay time of semi-synthetic aequorins prepared from coelenterazineanalogues 3a, 3e, 3f and 3j was markedly prolonged.

Example 3 Measurements of Luminescence Spectra of Semi-SyntheticAequorins

In a quartz cell with light path length of 10 mm, 1 ml of 50 mM Tris-HCl(pH 7.6) containing 1 mM EDTA and 100 μl (100 μg protein) of a solutionof the regenerated semi-synthetic aequorin were charged. Next, 100 μl of50 mM Tris-HCl (pH 7.6) containing 0.1 ml of 10 mM calcium chloridesolution was added thereto to start the luminescence reaction. Thespectra were measured on a spectrofluorimeter (FP-6500, manufactured byJasco Co., Ltd.) with the excitation light source turned off. Themeasurement conditions were bandwidth: 20 nm, response: 0.5 second, scanspeed: 2000 nm/min and 22 to 25° C. The luminescence spectra measuredwere corrected.

The results are summarized in TABLE 1. The results reveal that themaximum emission peaks of semi-synthetic aequorins prepared fromcoelenterazine analogues 3a, 3e, 3f and 3h were shifted toward thelonger wavelength by about 20 nm to 60 nm, by compared with the maximumemission peak at 472.5 nm from native aequorin prepared withcoelenterazine. On the other hand, it became clear that the maximumemission peaks of semi-synthetic aequorins prepared from coelenterazineanalogues 3b and 3j were shifted toward the shorter wavelength by about15 nm to 25.5 nm.

Furthermore, as shown in FIG. 1, it became clear that semi-syntheticaequorins prepared from coelenterazine analogues had wider spectra.

TABLE 1 Luminescence Properties of Semi-Synthetic Aequorins by Additionof Calcium Ions Lumi- Half Maximum Coelenterazine Luminescence nescencedecay luminescence analogue activity capacity time wavelength(abbreviation) I_(max) (%) 10 sec. (%) (sec) λ_(max) (nm) Coelenterazine100.0 100.0 0.81 472.5 (CTZ) 3a (TMD-296) 2.1 32.1 15.7 494.0 3b(TMD-282) 5.9 8.5 0.96 447.0 3d (TMD-276) 18.7 26.4 1.10 480.0 3e(TMD-277) 0.9 5.4 3.42 507.0 3f (TMD-278) 0.1 2.5 >60 515.0 3g (TMD-336)0.02 0.15 — — 3h (TMD-281) 2.1 6.5 1.71 532.5 3i (TMD-337) 0.3 1.3 — —3j (TMD-280) 4.2 67.0 16.8 457.5 (—) not measured (the activity is toolow)

Example 4 Measurements of Fluorescence Spectra of Novel FluorescentProteins Formed from Semi-Synthetic Aequorins

The fluorescence spectra of novel fluorescent proteins formed by theCa²⁺-triggered reaction from semi-synthetic aequorins in EXAMPLE 3 weremeasured at 25° C. in a quartz cell (10 mm light path length) using aJasco FP-6500 spectrofluorometer (excitation wavelength: 330 nm,emission/excitation bandwidth: 3 nm; response: 0.5 sec, scan speed, 1000nm/min). The fluorescence spectra measured were corrected.

The results are summarized in TABLE 2. It was revealed that all samplesmeasured showed flourescnece intensity. The fluorescence spectra areshown in FIG. 2. The fluorescence spectra of fluorescent proteinsobtained from coelenterazine analogues 3a, 3b, 3e, 3f, 3h and 3j weredifferent from the emission spectra of semi-synthetic aequorins.Especially with coelenterazine analogues 3b, 3e and 3j, the emissionspectra of semi-synthetic aequorins were markedly different from thefluorescence spectra of fluorescent proteins generated by the additionof calcium ions. This is the first example showing that the emissionspectra are different from the fluorescence spectra, in spite ofsemi-synthetic aequorins and fluorescent proteins from the samecoelenterazine analogues.

TABLE 2 Fluorescence spectra of fluorescent proteins Generated byAddition of Calcium Ions to Semi-Synthetic Aequorins Relative Maximumfluorescence fluorescence Coelenterazine analogue intensity wavelength(abbreviation) (rlu) λ_(max) (nm) Coelenterazine (CTZ) 63.3 473.0 3a(TMD-296) 118.1  501.5 3b (TMD-282) 10.3 580.0 3d (TMD-276) 61.8 482.53e (TMD-277) 28.0 468.5 3f (TMD-278) 38.5 525.0 3g (TMD-336) — — 3h(TMD-281) 58.7 537.0 3i (TMD-337) — — 3j (TMD-280) 13.3 580.0

Example 5 Preparation of Calcium Standard Solution

In 9 ml of 50 mM Tris-HCl (pH 7.6), 1 ml of 1 g/L calcium carbonatestandard solution (manufactured by Wako Pure Chemicals) was diluted toprepare 10⁻³ M calcium carbonate solution. One milliliter of theresulting 10⁻³ M calcium carbonate solution was taken and added to 9 mlof 50 mM Tris-HCl (pH 7.6) to prepare 10⁻⁴ M calcium carbonate solution.Next, 3 ml of the resulting 10⁻⁴ M calcium carbonate solution was takenand added to 6 ml of 50 mM Tris-HCl (pH 7.6) to prepare 3×10⁻⁴ M calciumcarbonate solution. One milliliter from the 10⁻⁴ M calcium carbonatesolution obtained was added to 9 ml of 50 mM Tris-HCl (pH 7.6) toprepare 10^(÷5) M calcium carbonate solution. Then, 3 ml of theresulting 10⁻⁵ M calcium carbonate solution was taken and added to 6 mlof 50 mM Tris-HCl (pH 7.6) to prepare 3×10⁻⁵ M calcium carbonatesolution. A dilution series was prepared by successively repeating theprocedures described above to give the 10⁻³ M to 10⁻⁸ M calcium standardsolutions.

Example 6 Preparation of Semi-Synthetic Aequorins for the Detection ofCalcium Concentration

In 5 ml of 50 mM Tris-HCl (pH 7.6) containing 10 mM DTT and 30 mM EDTA,5 mg of recombinant apoaequorin (manufactured by Chisso Corporation) wasdissolved and 100 μg of coelenterazine analogue dissolved in ethanol inan amount of 1.2-fold equivalent was added to the solution. The mixturewas allowed to stand at 4° C. overnight to convert into semi-syntheticaequorin. The resulting semi-synthetic aequorin was concentrated usingAmicon Ultra-4 (manufactured by Millipore, Inc., molecular weightcut-off: 10,000) to remove an excessive of coelenterazine analogue. Themixture was then washed 3 times with 3 ml of 30 mM Tris-HCl (pH 7.6)containing 0.05 mM EDTA to give the solution of semi-synthetic aequorincontaining 0.05 mM EDTA. This semi-synthetic aequorin solution (2.5mg/ml) was diluted with 20 mM Tris-HCl (pH 7.6) containing 0.1% BSA(manufactured by Sigma), 0.01 mM EDTA and 150 mM NaCl.

Example 7 Preparation of Calcium Standard Curve

The calcium standard solution prepared as above was dispensed into a96-well microplate (Nunc #236108) at 50 μl/well and 10 μl of the dilutedsemi-synthetic aequorin solution was injected into 50 μl of variousconcentration of calcium standard solution. The luminescence intensitywas measured for 60 seconds on a luminescence plate reader Centro LB960(manufactured by Berthold) and expressed in terms of the maximumintensity (I_(max)) of luminescence. The luminescence intensity wasmeasured on each semi-synthetic aequorin in the same procedures.Semi-synthetic aequorins for practically use were chosen by the maximumintensity (I_(max)) of luminescence and the calcium standard curve ofsemi-synthetic aequorin was prepared.

The results are shown in FIG. 3. As shown in FIG. 3, the calciumstandard curve can be prepared using semi-synthetic aequorins preparedfrom coelenterazine analogues (3a, 3b, 3d, 3e, 3f, 3h and 3j) of thepresent invention, indicating that the photoprotein of the presentinvention can be used for the detection, quantification and the like ofcalcium ions.

Example 8 Determination of Substrate Specificity and LuminescenceActivity of the 19 kDa Protein from Oplophorus luciferase

The 19 kDa protein from Oplophorus luciferase was purified by the methoddescribed in Inouye, S. and Sasaki, S., Protein Express. Purif. (2007)56: 261-268 and provided for use.

After 1 μl of the 19 kDa protein (2.3 mg/ml) from Oplophorus luciferasecontaining 1 mM DTT was dissolved in 100 μl of 30 mM Tris-HCl (pH 7.6)containing 10 mM EDTA, 1 μl of a solution of coelenterazine or itsanalogue (1 μg/μl) in ethanol was added to the solution to start theluminescence reaction, and the luminescence activity was measured for 60seconds on a luminometer luminescencer-PSN AB2200 (manufactured by AttoCo., Ltd.). The luminescence activity was determined by repeating themeasurement described above 3 times and expressed in terms of themaximum luminescence intensity (I_(max)) of luminescence activity.

The results are shown in TABLE 3. Coelenterazine analogue was not a goodsubstrate for Oplophorus luciferase.

TABLE 3 Luminescence activity of Oplophorus luciferase usingcoelenterazine analogues Coelenterazine analogue Luminescence(abbreviation) activity I_(max) (%) Coelenterazine (CTZ) 100.0 3a(TMD-296) 0.4 3b (TMD-282) 0.07 3d (TMD-276) 0.3 3e (TMD-277) 0.07 3f(TMD-278) 0.1 3g (TMD-336) 0.3 3h (TMD-281) 0.07 3i (TMD-337) 0.09 3j(TMD-280) 0.2

Example 9 Determination of Substrate Specificity and LuminescenceActivity of Gaussia luciferase

Gaussia luciferase was purified by the method described in JapanesePatent Application KOKAI No. 2008-099669 and provided for use. After 1μl of Gaussia luciferase (0.024 mg/ml) was dissolved in 100 μl ofphosphate buffered saline (manufactured by Sigma Inc.) containing 0.01%Tween 20 (manufactured by Sigma Inc.) and 10 mM EDTA, 1 μl of a solutionof coelenterazine or its analogue (1 μg/μl) in ethanol was mixed withthe solution to start the luminescence reaction. The luminescenceactivity was measured for 10 seconds on a luminometer luminescencer-PSNAB2200 (manufactured by Atto Co., Ltd.). The luminescence activity wasdetermined by repeating the measurement described above 3 times andexpressed in terms of the maximum luminescence intensity (I_(max)) ofluminescence activity.

The results are shown in TABLE 4. Coelenterazine analogue was not a goodsubstrate for Gaussia luciferase.

TABLE 4 luminescence activity of Gaussia luciferase using coelenterazineanalogues Coelenterazine Luminescence analogue (abbreviation) activityI_(max) (%) Coelenterazine (CTZ) 100.0 3a (TMD-296) 0.00 3b (TMD-282)0.01 3d (TMD-276) 0.00 3e (TMD-277) 0.00 3f (TMD-278) 0.00 3g (TMD-336)0.00 3h (TMD-281) 0.00 3i (TMD-337) 0.00 3j (TMD-280) 0.01

Example 10 Determination of Substrate Specificity and LuminescenceActivity of Renilla luciferase

Renilla luciferase was purified by the method described in Inouye, S. &Shimomura, O. Biochem. Biophys. Res. Commun. (1997) 233: 349-353, andused for assay.

After 1 μl of Renilla luciferase (0.45 mg/ml) was dissolved in 100 μl of30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA, 1 μl of a solution ofcoelenterazine or its analogue (1 μg/μl) in ethanol was added to thesolution to start the luminescence reaction. The luminescence activitywas measured for 10 seconds on a luminometer luminescencer-PSN AB2200(manufactured by Atto Co., Ltd.). The luminescence activity wasdetermined by repeating the measurement of luminescence activitydescribed above 3 times and expressed in terms of the maximumluminescence intensity (I_(max)) of luminescence activity.

The results are shown in TABLE 5. Coelenterazine analogue was not a goodsubstrate for Renilla luciferase.

TABLE 5 Luminescence activity of Renilla luciferase using coelenterazineanalogues Coelenterazine Luminescence analogue activity (abbreviation)I_(max) (%) Coelenterazine (CTZ) 100.0 3a (TMD-296) 0.00 3b (TMD-282)0.00 3d (TMD-276) 0.00 3e (TMD-277) 0.00 3f (TMD-278) 0.00 3g (TMD-336)0.00 3h (TMD-281) 0.00 3i (TMD-337) 0.00 3j (TMD-280) 0.03

Example 11 Preparation of Semi-Synthetic gFP from CoelenteramideAnalogues and Apoaequorin

Semi-synthetic gFP was prepared from recombinant apoaequorin andcoelenteramide analogues as follows. In 1 ml of 50 mM Tris-HCl (pH 7.6)containing 10 mM EDTA and 1 mM DTT, apoaequorin (0.2 mg) was mixed with8 μl of a coelenteramide analogue (1 μg/μl in anhydrous methanol). Themixture was allowed to stand 4° C. for 16 hours to preparesemi-synthetic gFP-like protein (hereinafter sometimes simply referredto as “semi-synthetic gFP”).

Example 12 Preparation of Semi-Synthetic BFP from CoelenteramideAnalogues and Apoaequorin

Semi-synthetic gFP was prepared from recombinant apoaequorin andcoelenteramide analogues as follows. In 1 ml of 50 mM Tris-HCl (pH 7.6)containing 10 mM CaCl₂ and 1 mM DTT, apoaequorin (0.2 mg) was mixed with8 μl of a coelenteramide analogue (1 μg/μl in anhydrous methanol). Themixture was allowed to stand at 4° C. for 16 hours to preparesemi-synthetic BFP-like protein (hereinafter sometimes simply referredto as “semi-synthetic BFP”).

Example 13 Measurement of the Fluorescence Spectra of CoelenteramideAnalogues, Semi-Synthetic BFP and Semi-Synthetic gFP

The fluorescence spectra were determined as follows. For coelenteramideanalogue alone, the fluorescence was measured according to theprocedures for semi-synthetic gFP and semi-synthetic BFP described inEXAMPLES 11 and 12, except for in the absence of apoaequorin, using 1 mlof 50 mM Tris-HCl (pH 7.6) containing 10 mM EDTA and 1 mM DTT or 1 ml of50 mM Tris-HCl (pH 7.6) containing 10 mM CaCl₂ and 1 mM DTT, in thefinal concentration of coelenteramide analogue adjusted to 8 μg/ml. Thefluorescence spectra were measured at 25° C. in a quartz cell (10 mmlight path length) using a Jasco FP-6500 spectrofluorometer (excitationwavelength: 337 nm, emission/excitation bandwidth: 3 nm; response: 0.5sec, scan speed, 1000 nm/min).

The results are shown in TABLES 6 and 7. In addition, the charts offluorescence spectra are shown in FIGS. 4 to 7.

Many of semi-synthetic BFPs and semi-synthetic gFPs prepared fromcoelenteramide analogues and apoaequorin showed much higher fluorescenceintensities than that of coelenteramide analogue alone. Also, most ofsemi-synthetic BFPs and semi-synthetic gFPs had different fluorescencespectra from the fluorescence spectra of coelenteramide analogue alone.

TABLE 6 Fluorescence spectra of coelenteramide analogues andsemi-synthetic gFPs in EDTA-containing buffer CoelenteramideSemi-synthetic gFP (EDTA-containing buffer) (EDTA-containing buffer)Maximum Maximum Coelenteramide fluorescence Fluorescence fluorescenceFluorescence analogue wavelength intensity wavelength intensity(abbreviation) λ_(max) (nm) (rlu) λ_(max) (nm) (rlu) Coelenteramide458.0 4.6 482.5 165.2 (CTMD) 4a (TMD-344) 471.0 1.9 521.5 82.9 4b(TMD-343) 495.5 7.1 530.0 43.2 4c (TMD-347) 475.5 11.8 499.0 32.1 4d(TMD-338) 462.5 12.4 504.5 58.8 4e (TMD-339) 478.0 28.9 487.0 72.6 4f(TMD-340) 448.5 2.3 536.5 70.5 4g (TMD-345) 473.0 5.5 512.5 108.5 4h(TMD-342) 426.5 115.2 426.5 68.9 544.0 70.0 4i (TMD-346) 471.5 12.1512.5 106.5 4j (TMD-341) 482.5 22.5 492.5 69.7 4k (TMD-373) 487.5 10.3532.0 139.9 4l (TMD-374) 523.5 9.3 524.5 140.5 4m (TMD-375) 429.0 9.8482.5 163.8 499.5 11.3 4n (TMD-376) N.D 485.5 151.5 4o (TMD-377) 482.52.1 485.0 3.4 4p (TMD-378) 476.0 5.3 523.0 21.1 4r (TMD-379) 481.0 10.3468.0 176.4 4s (TMD-348) 457.5 1.9 460.0 106.4 4t (TMD-349) 480.5 11.8482.5 22.0 4u (TMD-331) 440.5 0.7 494.5 11.4 4v (TMD-332) 484.0 1.4491.0 3.6 4w (TMD-330) 451.0 1.3 487.0 54.6 4x (TMD-365) 449.0 1.3 404.015.1 492.5 23.1 4y (TMD-366) 456.0 1.3 492.0 44.8 4z (TMD-368) 409.520.2 492.5 246.3 N.D. not detected

TABLE 7 Fluorescence spectra of coelenteramide analogues andsemi-synthetic BFPs in calcium ion-containing buffer CoelenteramideSemi-synthetic BFP (Buffer containing CaCl₂) (Buffer containing CaCl₂)Maximum Maximum Coelenteramide fluorescence Fluorescence fluorescenceFluorescence analogue wavelength wavelength wavelength wavelength(abbreviation) λ_(max) (nm) (rlu) λ_(max) (nm) (rlu) Coelenteramide457.0 5.9 468.5 151.2 (CTMD) 4a (TMD-344) 467.5 2.0 507.5 148.9 4b(TMD-343) 494.5 9.4 544.5 52.7 4c (TMD-347) 471.0 12.4 492.5 25.6 4d(TMD-338) 463.5 12.9 487.0 98.3 4e (TMD-339) 478.0 28.6 492.5 85.6 4f(TMD-340) 447.0 2.2 527.0 96.4 4g (TMD-345) 470.5 6.1 501.5 195.7 4h(TMD-342) 426.5 101.2 427.0 153.3 4i (TMD-346) 468.0 11.8 494.0 80.0 4j(TMD-341) 481.0 22.4 489.0 100.5 4k (TMD-373) 491.0 9.7 538.5 148.2 4l(TMD-374) 523.0 8.8 468.0 63.5 4m (TMD-375) 438.5 7.3 457.5 182.4 502.011.6 4n (TMD-376) N.D 487.5 231.3 4o (TMD-377) 478.5 2.1 477.5 3.9 4p(TMD-378) 484.5 5.4 548.5 70.0 4r (TMD-379) 483.0 10.2 468.0 159.9 4s(TMD-348) 468.5 2.0 408.5 22.1 4t (TMD-349) 482.0 12.1 479.5 29.5 492.041.5 4u (TMD-331) 445.5 0.9 402.0 6.1 487.5 5.2 4v (TMD-332) 466.0 1.4430.5 3.3 4w (TMD-330) 450.5 1.4 466.0 181.4 4x (TMD-365) 450.0 1.4485.0 36.8 4y (TMD-366) 457.0 1.4 405.5 15.8 482.5 12.6 4z (TMD-368)410.5 13.2 487.0 24.6 N.D. not detected

Example 14 Determination of the Luciferase Activity of Semi-SyntheticBFPs

To confirm that semi-synthetic BFPs prepared from coelenteramideanalogues and apoaequorin, which are novel fluorescent proteins havingluciferase activity, the luminescence reaction was performed in thepresence of calcium ions, using coelenterazine as a light emittingsubstrate. After 5 μl (corresponding to 1 μg protein) of a solution ofsemi-synthetic BFP prepared in EXAMPLE 12 was added to 100 μl of 50 mMTris-HCl (pH 7.6) containing 10 mM CaCl₂, 1 μl of a solution ofcoelenterazine (1 μg/μl) in ethanol was mixed with the solution to startthe luminescence reaction. The luminescence activity was measured for 10seconds on a luminometer luminescencer-PSN AB2200 (manufactured by AttoCo., Ltd.). The luminescence activity was determined by repeating themeasurement of luminescence activity described above twice and expressedin terms of the maximum luminescence intensity (I_(max)) of luminescenceactivity. The results are shown in TABLE 8.

It became clear that semi-synthetic BFP has a sufficient luciferaseactivity. That is, semi-synthetic BFP was able to prepare variousfluorescent proteins having the fluorescence activity and luciferaseactivity.

TABLE 8 Luciferase activity of semi-synthetic BFP using coelenterazineas a substrate Coelenteramide analogue Luciferase activity used toprepare of semi-synthetic semi-synthetic BFP BFP (%) CTMD 100.0 4a(TMD-344) 80.0 4b (TMD-343) 104.3 4c (TMD-347) 79.5 4d (TMD-338) 104.14e (TMD-339) 75.2 4f (TMD-340) 40.0 4g (TMD-345) 69.1 4h (TMD-342) 107.84i (TMD-346) 89.8 4j (TMD-341) 71.3 4k (TMD-373) 39.0 4l (TMD-374) 75.44m (TMD-375) 42.2 4n (TMD-376) 147.4 4o (TMD-377) 119.5 4p (TMD-378)159.7 4r (TMD-379) 100.4 4s (TMD-348) 96.8 4t (TMD-349) 90.0 4u(TMD-331) 89.3 4v (TMD-332) 96.8 4w (TMD-330) 65.9 4x (TMD-365) 122.9 4y(TMD-366) 125.5 4z (TMD-368) 121.6

[Sequence Listing Free Text] [SEQ ID NO: 1]

This is the nucleotide sequence of native apoaequorin.

[SEQ ID NO: 2]

This is the amino acid sequence of native apoaequorin.

[SEQ ID NO: 3]

This shows the nucleotide sequence of native apoclytin-I.

[SEQ ID NO: 4]

This shows the amino acid sequence of native apoclytin-I.

[SEQ ID NO: 5]

This shows the nucleotide sequence of native apoclytin-II.

[SEQ ID NO: 6]

This shows the amino acid sequence of native apoclytin-II.

[SEQ ID NO: 7]

This shows the nucleotide sequence of native apomitrocomin.

[SEQ ID NO: 8]

This shows the amino acid sequence of native apomitrocomin.

[SEQ ID NO: 9]

This shows the nucleotide sequence of native apobelin.

[SEQ ID NO: 10]

This shows the amino acid sequence of native apobelin.

[SEQ ID NO: 11]

This shows the nucleotide sequence of native apobervoin.

[SEQ ID NO: 12]

This shows the amino acid sequence of native apobervoin.

1-3. (canceled)
 4. A compound represented by general formula (II) below;

wherein R^(3′) is hydrogen atom, bromine atom and any one selected fromthe groups represented by formulas below:

wherein each of R^(6′), R^(7′) and R^(8′) independently representshydrogen atom, a substituted or unsubstituted alkyl having 1 to 6 carbonatoms, a substituted or unsubstituted aryl, or a substituted orunsubstituted heteroaryl; and R^(6′) and R^(8′) may be combined togetherto form a substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl with the carbon atom bound to each of R^(6′)and R^(8′).
 5. The compound according to claim 4, which is selected fromthe compounds below:


6. A compound represented by general formula (III) below:

wherein Z¹ is O or S; and R^(3″) is hydrogen atom, bromine atom, asubstituted or unsubstituted aryl, a substituted or unsubstitutedarylalkyl, a substituted or unsubstituted arylalkenyl, a substituted orunsubstituted arylalkynyl, an alkyl which may optionally be substitutedwith an alicyclic group, an alkenyl which may optionally be substitutedwith an alicyclic group, an alkynyl which may optionally be substitutedwith an alicyclic group, an alicyclic group, or a heterocyclic group. 7.A compound represented by general formula (IV) below:

wherein R^(2′″) is a group selected from the groups below:

and R^(3′″) is hydrogen atom, bromine atom, a substituted orunsubstituted aryl, a substituted or unsubstituted arylalkyl, asubstituted or unsubstituted arylalkenyl, a substituted or unsubstitutedarylalkynyl, an alkyl which may optionally be substituted with analicyclic group, an alkenyl which may optionally be substituted with analicyclic group, an alkynyl which may optionally be substituted with analicyclic group, an alicyclic group, or a heterocyclic group.
 8. Thecompound according to claim 6, which is selected from the compoundsbelow:

9-12. (canceled)
 13. A fluorescent protein comprising the compoundaccording to claim 4, an apoprotein of a calcium-binding photoproteinand a calcium ion or a divalent or trivalent ion replaceable for thecalcium ion.
 14. (canceled)
 15. A method for producing a fluorescentprotein, which comprises contacting the compound according to claim 4with an apoprotein of a calcium-binding photoprotein in the presence ofa calcium ion or a divalent or trivalent ion replaceable for the calciumion to obtain the fluorescent protein.
 16. (canceled)
 17. A fluorescentprotein comprising the compound according to claim 4 and an apoproteinof a calcium-binding photoprotein.
 18. A method for producing afluorescent protein, which comprises contacting the compound accordingto claim 4 with an apoprotein of a calcium-binding photoprotein in thepresence of a chelating agent for removing a calcium ion or a divalentor trivalent ion replaceable for the calcium ion to obtain thefluorescent protein.
 19. A method for producing a fluorescent protein,which comprises treating the fluorescent protein according to claim 13with a chelating agent for removing a calcium ion or a divalent ortrivalent ion replaceable for the calcium ion.
 20. The method accordingto claim 18, wherein the contact is carried out in the presence of areducing agent.
 21. A method for analyzing a physiological function orenzyme activity, which comprises performing the fluorescence resonanceenergy transfer (FRET) method using the fluorescent protein according toclaim 13 as an acceptor or a donor.